FIELD

[0001] The present disclosure relates to the field of molecular biology. More particularly, it relates to methods and compositions useful for the detection, amplification, and quantification of nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATIONS AND INCORPORATION OF

SEQUENCE LISTING

[0002] This application claims the benefit of U.S. Provisional Patent Application No. 63/131,747, filed December 29, 2020, which is incorporated by reference herein in its entirety. The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on December 8, 2021, is named P35027WO00_SL.txt, and is 36,864 bytes in size measured in Microsoft Windows®.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0003] This invention was made with United States Government support under Grant No. R01CA203964 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND

[0004] Polymerase chain reaction (PCR) is widely used to quickly generate a large number of targeted DNA template copies. The resulting products can be used for further sequencing and detecting DNA variants. To ensure successful production of the correct target segments, two primers specifically designed to bind to the breakpoints of target DNA segments are required. Thus, for an efficient PCR amplification, two separate target DNA sequences that are intended to bind to the primers must be conserved.

[0005] However, there are many possible situations in which one or both of the targeted primer binding site sequences may be altered, resulting in a negative PCR amplification reaction. For example, viruses mutate rapidly, leading to DNA mutations in the primer binding regions. In cancer cells, chromosomes may exhibit large scale DNA structural variants (e.g., fusions and translocations).

[0006] Significant needs remain to develop assays for detecting large-scale DNA structural variants, especially for selectively enriching and detecting DNA variants with a low fraction or frequency.

SUMMARY

[0007] In an aspect, this application provides a method of preparing a nucleic acid template into a DNA library for sequencing, the method comprising the steps of: (a) Mixing a nucleic acid template, a Ligation Tail Adapter (LTA) molecule, a DNA ligase, and reagents for DNA ligase activity to form a first mixture; where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand which two strands form a Double-Stranded End (DSE) and a DNA Tail (DT) region; (b) Subjecting the first mixture to a suitable temperature to allow for DNA ligation to form a ligation product mixture; (c) Introducing to the ligation product mixture from step (b) a targetspecific Outer Forward Primer (OFP) and a Splint, a DNA polymerase, and reagents for DNA polymerase activity to form a second mixture; where the OFP comprises a nucleic acid sequence that can specifically anneal to a portion of a target DNA sequence region of interest, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region or portion thereof; (d) Subjecting the second mixture to a suitable temperature to allow for DNA polymerase extension to form a DNA polymerase extension product mixture; (e) Introducing to the DNA polymerase extension product mixture from step (d) a target-specific Inner Forward Primer (IFP), a Universal Primer (UP), a thermostable DNA polymerase, and reagents for DNA polymerase activity to form a third mixture, where the IFP comprises a nucleic acid sequence that can specifically anneal to a portion of the target DNA sequence region of interest; and where the UP comprises a sequence which can specifically anneal to the 5' end subsequence, or portion thereof, of the Splint; and (I) Subjecting the third mixture to temperature cycles to allow for polymerase chain reaction (PCR) amplification. [0008] In an aspect, this application provides a method for preparing a nucleic acid for sequencing, the method comprising: (i) ligating a Ligation Tail Adapter (LTA) molecule to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand, and where the LTA- top strand and the LTA-bottom strand form a Double-Stranded End (DSE) and a DNA Tail (DT) region to produce a ligation product; (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a Splint, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region to produce an amplification product; and (iii) amplifying an amplification product using a second target-specific primer that specifically anneals to the amplification product and a Universal Primer (UP) comprising a sequence which can specifically anneal to the 5' end subsequence, or a portion thereof, of the Splint, where the second target-specific primer is nested relative to the first target-specific primer. [0009] In an aspect, this application provides a method for preparing a nucleic acid for sequencing, the method comprising: (i) ligating a Ligation Tail Adapter (LTA) molecule to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand, and where the LTA- top strand and the LTA-bottom strand which two strands form a Double-Stranded End (DSE) and a DNA Tail (DT) region to produce a ligation product; (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a Splint, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region to produce an amplification product; and (iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a Universal Primer (UP) comprising a sequence which can specifically anneal to the 5' end subsequence, or a portion thereof, of the Splint, where the second target-specific primer is nested relative to the first target-specific primer.

[0010] In an aspect, this application provides a method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising: (a) ligating a target nucleic acid molecule comprising the known target nucleotide sequence with a universal Ligation Tail Adapter (LTA), where the universal LTA comprises a non-amplification strand and an amplification strand to produce a ligation product; (b) amplifying a portion of the target nucleic acid molecule and the amplification strand of the universal LTA with a Splint and a first targetspecific primer from the ligation product to produce a first amplicon; (c) amplifying a portion of the first amplicon with a Universal Primer (UP) and a second target-specific primer to produce a second amplicon; and (d) sequencing the second amplicon using a first sequencing primer and a second sequencing primer; where the universal LTA comprises a ligatable Double-Stranded End (DSE) and a DNA Tail (DT) region; where the non-amplification strand comprises a 5' duplex portion; where the amplification strand comprises an unpaired 5' portion, a 3' duplex portion, and a 3' thymine (T) overhang; where the duplex portion of the non-amplification strand and the duplex portion of the amplification strand are complementary and form the ligatable DSE comprising a 3' T overhang; where the duplex portion is of sufficient length to remain in duplex form at the ligation temperature; where the first target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid molecule; where the second target-specific primer comprises a 3' portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence within the first amplicon, and a 5' portion comprising a nucleic acid sequence that is identical to the second sequencing primer and the second target-specific primer is nested with respect to the first targetspecific primer.

[0011] In an aspect, this application provides a method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising: (i) ligating a universal Ligation Tail Adapter (LTA) to a nucleic acid molecule comprising the known target nucleotide sequence to produce a ligation product; (ii) amplifying the ligation product via polymerase chain reaction using a Splint that specifically anneals to the universal LTA, and a first target-specific primer that specifically anneals to the known target nucleotide sequence to produce a first amplification product; (iii) amplifying the first amplification product via polymerase chain reaction using a Splint-specific primer and a second target-specific primer, where the second target-specific primer is nested relative to the first target-specific primer to produce a second amplification product; and (iv) sequencing the second amplification product using a first sequencing primer and a second sequencing primer, where the first sequencing primer and the second sequencing primer are complementary to opposite strands of the second amplification product.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1: A schematic illustration of an example of adjacent nested PCR reaction scheme using LTAs. LTA molecules are ligated to template DNA molecules. The half arrows in the figure indicate the 3' end of a DNA strand. Letter P outlined with a circle means this DNA base is modified with a phosphate. Letter ‘T’ indicates a T base overhang. An LTA molecule comprises a LTA-top strand and a LTA-bottom strand. The DSE is a double-stranded DNA. Two PCR steps are shown. The first PCR step (an outer PCR step) is using an OFP as a forward primer and a Splint as a reverse primer. The OFP has over 90% percent complementarity within a binding site of the template sequence and the Splint has over 90% percent complementarity within a binding site in the reverse complementary strand of the LTA-top strand. The second PCR step (an inner PCR step) uses an IFP as a forward primer and an UP as a reverse primer. The IFP has over 90% percent complementarity within a binding site in the template in the 3' downstream of the region relative to the OFP binding site. The UP has over 90% percent complementarity within a binding site in the Splint strand. The amplicon is obtained and named as Nested PCR Amplicon.

[0013] Figure 2: Schematic illustration of exemplary structures of the LTA. (a) An LTA comprises a double-stranded DNA DSE and a double-stranded DT region with a single-stranded UMI loop between the DSE and the DT region, (b) An LTA comprises a DSE with a length of 5 - 50 bp. The LTA-top strand comprises a 3' overhang which cannot bind to the LTA-bottom strand. In addition, the LTA-bottom strand does not contain any single strand DNA that can bind to the LTA-top strand, (c) The structure is similar to the structure of (b) but with a Unique Molecular Identifier (UMI) or sample barcode (SB) between the DSE and the 5' overhang of the LTA-top strand, (d) An LTA comprises an LTA-top strand and an LTA-bottom strand which can form a DSE region. Alternatively, the LTA-bottom strand comprises a 5' overhang that cannot bind to the LTA-top strand and in turn constitutes a DT region, (e) The structure is similar to the structure of (c) but with a UMI or an SB between the double-stranded DNA part and the 5' overhang of the LTA-bottom strand. (1) An LTA comprises an LTA-top strand and an LTA-bottom strand which can form a DSE region. Alternatively, the LTA-top strand comprises the 3' overhang that cannot bind to LTA-bottom strand. In addition, the LTA-bottom strand comprises a 5' overhang that cannot bind to the LTA-top strand, (g) The structure is similar to the structure of (1) but with a UMI or an SB between the DSE and the 5' overhang of the LTA-bottom strand, (h) The structure is similar to the structure of (1) but with a UMI or an SB between the DSE and the 3' overhang of the LTA-top strand.

[0014] Figure 3: Exemplary UMI composition with bases of 8 used in an LTA molecule, (a) An UMI sequence is added between the DSE and the DT region, (b) The UMI can be a mixture of fixed UMIs with pairwise Hamming Distance minimum. For example, UMI 1 and UMI 2 have two different bases in position 2 and position 5, and other bases are the same. As another example, UMI 2 and UMI 3 have two different bases in position 2 and position 7, and other bases are the same, (c) The UMI can be a mixture of fixed UMIs with pairwise Levenschtein Distance minimum. The UMI 1 and UMI 2 have 8 bases and they can match with each after filling in one base gap. UMI 1 and UMI 2 have the same bases in positions 1, 3, 4, 5, 6, 7, and 9, UMI 1 position 2 matches with a gap in UMI 2, and UMI 2 position 8 matches with a gap in UMI 1. (d) The Randomer UMI that can be H and/or G compositions.

[0015] Figure 4: Exemplary schematic illustration of the binding structures of a Splint and an LTA-top strand. A Splint can have a 5' overhang that cannot bind to the LTA-top strand with a length of 1 - 100 nt. (a) Splint- 1 can bind to the single-stranded DNA of the LTA-top from the second base following the DSE region to the 3' end of the LTA-top strand, where the binding region has a length of, for example, 10 nucleotides, (b) Splint-2 has a 3' region which is reverse complementary to the entire DT region of the LTA-top strand and have a 5' region overhang, (c) The 3' end of the Splint-3 binds to the LTA-top strand starting from second base of the 5' end to the second base of the LTA-top strand in DSE region 3' end. (d) The 3' end of Splint-4 binds to the 5' end of the DSE region of the LTA-top strand, (e) The 5' end of Splint-5 comprises a hairpin structure.

[0016] Figure 5: Exemplary schematic illustration of the binding positions of the OFP and IFP. An OFP and an IFP can each have over 90% percent identity or complementarity to the reverse complementary strand of the template. The IFP binding position is generally in the 5' upstream compared to the OFP binding position, (a) The 5' end of an IFP tiles to the 3' end of an OFP. (b) The 5' end of an IFP is located at a distance of about 1 - 50 nt away from the 3' end of an OFP. (c) The 5' end of an IFP has a 1 - 40 bp overlap with the 3' end of an OFP.

[0017] Figure 6: Exemplary schematic illustration of a UP binding to a Splint. The UP-1 sequence comprises a sequence that is over 70% homologous to the reverse complementary sequence of the 5' overhang of the Splint. The 3' end of the UP-1 starts from the second base to the end of the 5' overhang of the Splint. The UP-1 has a single-stranded DNA oligo that cannot bind to the Splint. The UP -2 has over 90% percent identity to the reverse complement of the Splint 5' overhang. The UP-3 has over 90% percent identity to the reverse complement of the Splint where the 3' end of UP-3 matches the 5' end of the single-stranded DNA part of the LTA and the 5' end of UP-3 matches the 3' end of the Splint.

[0018] Figure 7: Exemplary schematic illustration of a nested PCR amplicon used for NGS library preparation. Nested PCR amplicon is used in this example as an insert template for an adapter PCR. The inner AD-FP (adaptor forward primer) comprises an adapter sequence and the IFP sequence. The UP primer acts as the reverse primer and undergoes a 2-cycle PCR for adding the adapter to the template. This step also can be merged with the Figure 1 inner PCR step (i.e., the PCR step with the UP and the IFP). An index PCR follows using Illumina sequencing index primer P5 as forward primer and P7 as reverse primer. The final products can be sequenced by the Illumina platform using the pair end sequencing method.

[0019] Figure 8: Exemplary schematic illustration of a design scheme for detecting unknown FGFR2 RNA fusions. The IFP here is shown to have a 5-20 nt gap to the breakpoint. The OFP binds to the upstream strand of the known partner. [0020] Figure 9: Exemplary reads distribution of WT library and fusion library align to genome reads distribution. All the reads are trimmed with a sequencing adapter, aligned to an Hgl 9 reference genome, and filtered out the reads that are shorter than 50 bp. (a) Reads distribution is aligned to the gene FGFR2. The reads are shown in loglO scale and the axis is from 0 to 400,000. The reads distribute in exon 16, 17, 18, and 19. (b) The fusion reads are found in exon 3 of the gene BICC1 which is from gBlock2. (c) the fusion reads are aligned to exon 2 of gene GAB2 and (d) the fusion reads are aligned to exon 5 and exon 6 of the gene AHCYL1.

[0021] Figure 10: Exemplary NGS results from RNA fusion detection. RNA reference material is ordered from SeraCare. The RNA reference material contains a NTRK1 gene fusion and three other gene exons as fusion partners. All the three gene fusion partners are found from (a) to (c). NTRK1 gBlocks is mixed with NA18537 (control sample) as the control sample which contains the LMNA exon 2 sequence but not include TPM3 exon 7 partner or TGF exon 5 partner. In panel (a), the reads are mapped to TPM3 exon 7 in SeraCare RNA samples but not so in the control sample, since the control sample does not have this fusion partner. The gBlocks mixed with NA18537 does not detect any reads, since there is no fusion template in TPM3 genes. Panel (b) shows the reads are in TGF exon 5 in SeraCare sample but not in the control sample since control sample doesn’t have this fusion partner. In panel (c), the LMNA exon 2 reads are shown in both SeraCare RNA and the control sample, since both of them have this fusion partner. All the fusion partners are detected in the reference RNA sample.

[0022] Figure 11: Exemplary schematic illustration of design schemes for detecting unknown DNA fusion, (a) An unknown DNA fusion with a known exon on one side, the design targets on the intron region which is in the 3' downstream of the known exon. One design group contains an OFP and an IFP to target one region. The gap between different groups ranges from 0 nt to 100 nt. The groups will tile the whole intron region, (b) The known exon can be in the fusion downstream partner, so that we can design primers to target the 5' intron and target in the negative strand.

[0023] Figure 12: Exemplary DNA fusion reads from NGS results. The aH2228 sample has been validated to have an EML4-ALK fusion. Using our ligation tail adapter approach, we also find fusion reads with a breakpoint in EML4 intron 6 and ALK intron 19. The WT reads only contain the ALK intron 19 sequence.

[0024] Figure 13: Exemplary schematic illustration of ABDA workflow for fusion detection. The use of LTA and Splint molecules are essentially as illustrated in Figure 1. The left side workflow shows the ABDA for WT detection. The Blocker will bind to the WT template. The IFP cannot bind to the template and will inhibit the PCR reaction. On the right side, when the template comprises a gene fusion sequence, the Blocker cannot bind to the template perfectly, and the IFP can displace the Blocker so that the fusion template can be enriched via a PCR reaction. After the inner PCR step, the sequencing adapter is added to the amplicons through two cycles of PCR.

[0025] Figure 14: Exemplary schematic illustration of having UMI in the LTA-bottom strand. In the first cycle, an OFP amplifies the template, and a UMI or SB are added to the DNA template positive strand. Then, the OFP functions as a forward primer and the Splint as a reverse primer in the first PCR reaction. As an optional further step, an IFP as a forward primer and a non- complementary portion of the Splint as a reverse primer are used to amplify for several cycles. In this optional step, the reverse primer can further include a sequencing adapter primer sequence for adding sequencing index.

[0026] Figure 15: Exemplary on-target rate of Y adapter and Ligation Tail adapter. Five primers are designed targeting on different exons of the FGFR2 gene. On-target-rate values are compared between Y adapter and LTA. Three different reverse primers of UP-1, UP -2, and UP-3 (see Figure 6) are used for PCR amplification. All three different reverse primers achieve higher on-target-rate than Y adapters.

DETAILED DESCRIPTION

[0027] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York).

[0028] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0029] Any composition provided herein is specifically envisioned for use with any applicable method provided herein. [0030] When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc.

[0031] The term “and/or” when used in a list of two or more items means any one of the listed items by itself or in combination with any one or more of the other listed items. For example, the expression “A and/or B” is intended to mean either or both of A and B - i.e., A alone, B alone, or A and B in combination. The expression “A, B and/or C” is intended to mean A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B, and C in combination.

[0032] When a range of numbers is provided herein, the range is understood to inclusive of the edges of the range as well as any number between the defined edges of the range. For example, “between 1 and 10” includes any number between 1 and 10, as well as the number 1 and the number 10.

[0033] As used herein, the singular form “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a compound” or “at least one compound” may include a plurality of compounds, including mixtures thereof. As used herein, the term “plurality” refers to any number greater than one.

[0034] As used herein, “approximately,” in reference to a number, means plus and/or minus 10% of that number. For example, “approximately 100” means anywhere from 90 to 110.

[0035] As used herein, the term “Ligation Tail Adapter”, “LTA”, or “Ligation Tail Adapter (LTA) molecule” refers to a nucleic acid molecule comprised of two strands (referenced as an LTA-top strand and an LTA-bottom strand, or alternatively, an amplification strand and a nonamplification strand) and comprising a first end which is a ligatable duplex end (Double-Stranded End (DSE)) and a second end having a DNA Tail (DT) region. An amplification strand and a nonamplification strand differ in that the former (or portion thereol) is included in an amplification product, while the latter is not. In an aspect, an LTA-top strand or a non-amplification strand comprises a 5 ' duplex portion. In another aspect, an LTA-top strand or an amplification strand comprises an unpaired 5 ' portion, a 3 ' duplex portion, and a 3 ' T overhang and nucleic acid sequences identical to a first and second sequencing primers. In an aspect, the duplex portions of an LTA-top strand and an LTA-bottom strand are substantially complementary and form the first ligatable duplex end (e.g., DSE) comprising a 3 ' T overhang and the duplex portion is of sufficient length to remain in duplex form at the ligation temperature. In an aspect, an LTA-top strand comprises a phosphorylated nucleotide at its 5' end. In an aspect, an LTA-bottom strand comprises a thymine at its 3' end. In an aspect, an LTA-top strand comprises a phosphorylated nucleotide at its 5' end, and an LTA-bottom strand comprises a thymine at its 3' end. In an aspect, the second nucleotide position from the 3' end of an LTA-bottom strand comprises a phosphorothioate bond modification.

[0036] As used herein, a “DNA Tail” or “DT” region refers to a 3' region of an LTA-top strand or an LTA-bottom strand. In an aspect, a DT region comprises a single-stranded DNA. In an aspect, a DT region comprises a double-stranded DNA. In an aspect, a DT region comprises a doublestranded sequence comprising between 1 and 30 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 1 and 25 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 1 and 20 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 1 and 15 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 1 and 10 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 1 and 5 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 5 and 10 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 10 and 15 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 15 and 20 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 20 and 25 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 25 and 30 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 5 and 25 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 10 and 20 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 5 and 30 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 10 and 30 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 15 and 30 nucleotide mismatches. In an aspect, a DT region comprises a double-stranded sequence comprising between 20 and 30 nucleotide mismatches.

[0037] As used herein, a “Double-Stranded End” or “DSE” refers to a region of doublestranded DNA formed between an LTA-top strand and an LTA-bottom strand. In an aspect, the forward strand of a DSE begins from the 5' end of an LTA-top strand, and the reverse strand of the DSE begins from the second nucleotide position from the 3' end of an LTA-bottom strand. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 90 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 80 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 70 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 60 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 50 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 40 nucleotides. In an aspect, a DSE is a doublestranded DNA molecule comprising a length of between 5 nucleotides and 30 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 20 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 5 nucleotides and 10 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 10 nucleotides and 20 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 15 nucleotides and 25 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 20 nucleotides and 30 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 25 nucleotides and 35 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 30 nucleotides and 40 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 35 nucleotides and 45 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 40 nucleotides and 50 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 45 nucleotides and 55 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 50 nucleotides and 60 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 55 nucleotides and 65 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 60 nucleotides and 70 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 65 nucleotides and 75 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 70 nucleotides and 80 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 75 nucleotides and 85 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 10 nucleotides and 80 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 15 nucleotides and 70 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 20 nucleotides and 60 nucleotides. In an aspect, a DSE is a double-stranded DNA molecule comprising a length of between 25 nucleotides and 50 nucleotides.

[0038] In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is at least 90%. In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is at least 92.5%. In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is at least 95%. In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is at least 97.5%. In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is at least 99%. In an aspect, the percent identity between a LTA-top strand and the reverse complementary of the LTA-bottom strand in a DSE region is 100%.

[0039] As used herein, a “Splint” refers to a nucleic acid molecule that is capable of bridging together two nucleic acid molecules (e.g, a first nucleic acid molecule and a second nucleic acid molecule) that do not share sequence complementarity and are not normally physically linked to each other. Typically, a Splint comprises at least 90% complementarity to a first nucleic acid molecule and at least 90% complementarity to a second nucleic acid molecule. In an aspect, a Splint is a primer.

[0040] In an aspect, a Splint comprises a 5' sequence that does not bind with an LT A. In an aspect, a Splint comprises a 5' sequence that comprises between 1 nucleotide and 100 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 3 nucleotides and 5 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 5 nucleotides and 7 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 7 nucleotides and 10 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 10 nucleotides and 15 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 15 nucleotides and 20 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 20 nucleotides and 25 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 25 nucleotides and 30 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 35 nucleotides and 40 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 40 nucleotides and 45 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 45 nucleotides and 50 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 50 nucleotides and 55 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 55 nucleotides and 60 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 60 nucleotides and 65 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 65 nucleotides and 70 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 70 nucleotides and 75 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 75 nucleotides and 80 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 80 nucleotides and 85 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 85 nucleotides and 90 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 90 nucleotides and 95 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 90 nucleotides and 100 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 95 nucleotides and 100 nucleotides.

[0041] In an aspect, a Splint comprises a 5' sequence that comprises between 3 nucleotides and 10 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 5 nucleotides and 15 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 10 nucleotides and 20 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 15 nucleotides and 25 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 20 nucleotides and 30 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 25 nucleotides and 35 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 35 nucleotides and 45 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 5 nucleotides and 25 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 15 nucleotides and 50 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 20 nucleotides and 55 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 35 nucleotides and 60 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 50 nucleotides and 75 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises between 65 nucleotides and 90 nucleotides.

[0042] In an aspect, a Splint comprises a 5' sequence that comprises at least 3 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 5 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 7 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 10 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 15 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 20 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 25 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 30 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 35 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 40 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 45 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 50 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 55 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 60 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 65 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 70 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 75 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 80 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 85 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises at least 90 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 3 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 5 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 7 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 10 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 15 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 20 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 25 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 30 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 35 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 40 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 45 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 50 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 55 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 60 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 65 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 70 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 75 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 80 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 85 nucleotides. In an aspect, a Splint comprises a 5' sequence that comprises less than or equal to 90 nucleotides.

[0043] In an aspect, a Splint comprises a 3' sequence capable of binding to a single-stranded 3' overhang of an LTA-top strand from the second nucleotide position following the DSE to at least the 10 ^th nucleotide position on the 3' end of a DT region.

[0044] In an aspect, a Splint comprises a 3' sequence capable of binding to a 3'overhang of an LTA-top strand from the first nucleotide position following a DSE. [0045] In an aspect, a Splint comprises a 3' sequence capable of binding to a DSE region starting from the second nucleotide position on the 5' end of an LTA-top strand to the second nucleotide position of the 3' end of the DSE.

[0046] In an aspect, a Splint comprises a 3' region capable of biding to a DSE region starting from the first nucleotide position of the 5' region of an LTA-top strand.

[0047] In an aspect, a Splint binds to an LTA-bottom strand instead of an LTA-top strand.

[0048] In an aspect, a Splint comprises, in order from 5' to 3', a first sequence, a second sequence, a third sequences, and a fourth sequence, where the third sequence is complementary to the first sequence.

[0049] As used herein, in reference to a Splint, a “5' sequence” refers to a sequence in the 5’ half of the Splint, and a “3' sequence” refers to a sequence in the 3' half of the Splint (with each half being based on the number of nucleotides in the Splint).

[0050] In an aspect, the 3' end subsequence of a Splint can specifically anneal to a DT region or a portion of the DT region.

[0051] In an aspect, an LTA-top strand comprises a single-stranded DT region that does not bind to an LTA-bottom strand. In an aspect, an LTA-bottom strand 5' region binds to an LTA-top strand to form a DSE. In an aspect, an LTA-top strand comprises a single-stranded DT region that does not bind to an LTA-bottom strand and an LTA-bottom strand 5' region binds to an LTA-top strand to form a DSE.

[0052] In an aspect, an LTA-bottom strand comprises a single-stranded DT region that does not bind to an LTA-top strand. In an aspect, an LTA-top strand 3' region binds to an LTA-bottom strand without any overhanging nucleotides to form a DSE. In an aspect, an LTA-bottom strand comprises a single-stranded DT region that does not bind to an LTA-top strand, and the LTA-top strand 3' region binds to an LTA-bottom strand without any overhanging nucleotides to form a DSE.

[0053] In an aspect, both an LTA-top strand 3' region and an LTA-bottom strand 5' region comprise a single-stranded DT region that does not bind to each other, and where the LTA-top strand 5' region and the LTA-bottom strand 3' region bind with each other to form a DSE.

[0054] In an aspect, an LTA-top strand comprises a single-stranded UMI between a DSE and a DT region. In an aspect, an LTA-top strand comprises an SB between a DSE and a DT region. In an aspect, an LTA-top strand comprises a single-stranded UMI between a DSE and a DT region and an SB between the DSE and the DT region. In an aspect, an LTA-bottom strand comprises a single-stranded UMI between a DSE and a DT region. In an aspect, an LTA-bottom strand comprises an SB between a DSE and a DT region. In an aspect, an LTA-bottom strand comprises a single-stranded UMI between a DSE and a DT region and an SB between the DSE and the DT region.

[0055] In an aspect, both an LTA-top strand and an LTA-bottom strand comprise a singlestranded UMI between a DSE and a DT region. In an aspect, both an LTA-top strand and an LTA- bottom strand comprise an SB between a DSE and a DT region. In an aspect, both an LTA-top strand and an LTA-bottom strand comprise a single-stranded UMI between a DSE and a DT region and an SB between the DSE and the DT region.

[0056] As used herein, a “5' region” refers to the first 25% of the nucleotides of a given nucleic acid molecule when counting from 5' to 3' when starting from the 5'-most nucleotide. When a 5' region of a first nucleic acid molecule binds to a second nucleic acid molecule, it will be appreciated that the entire 5' region of the first nucleic acid molecule does not have to bind to the second nucleic acid molecule (e.g, partial binding is specifically envisioned). Similarly, as used herein, a “3' region” refers to the last 25% of the nucleotides of a given nucleic acid molecule when counting from 5' to 3' when starting from the 5'-most nucleotide. When a 3' region of a first nucleic acid molecule binds to a second nucleic acid molecule, it will be appreciated that the entire 3' region of the first nucleic acid molecule does not have to bind to the second nucleic acid molecule (e.g, partial binding is specifically envisioned).

[0057] The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or amino acid sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or amino acid) over a window of comparison (the “alignable” region or regions), (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins and polypeptides) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%.

[0058] The terms “percent complementarity” or “percent complementary” as used herein in reference to two nucleotide sequences refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e. , without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of complementary nucleotide bases, such as guanine (G)-cytosine (C), adenine (A)-thymine (T), and A-uracil (U), through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base-paired nucleotides), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

[0059] As used herein, a “portion” of a nucleic acid molecule refers to contiguous set of nucleotides comprised by that molecule. A portion can comprise all or only a subset of the nucleotides comprised by the molecule. A portion can be double-stranded or single-stranded.

[0060] As used herein, a “nucleotide mismatch” refers to an alignment of two sequences that pairs two uncomplimentary nucleotides. Non-limiting examples of mismatches include G-A, G-T, G-U, G-G, C-A, C-T, C-U, C-C, A-A, T-T, and T-U. Conversely, “matched” alignments of nucleotides refer to complimentary pairs such as G-C, A-T, and A-U.

[0061] As a non-limiting example, the complement of the sequence 5'-ATGC-3' is 3'-TACG- 5', and the reverse complement of 5'-ATGC-3' is 5'-GCAT-3'. Notably, the complement and reverse complement sequences are identical to each other when viewed in the 5' to 3' direction.

[0062] For optimal alignment of sequences to calculate their percent complementarity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool® (BLAST™), etc., that can be used to compare the sequence complementarity or identity between two or more nucleotide sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm, see, e.g., Chenna etal., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31 : 3497-3500 (2003); Thompson et al. , “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); Larkin MA et al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul et al. "Basic local alignment search tool." J. Mol. Biol. 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference. For alignment between fusion sequences, alternative programs such as STAR or STAR- Fusion can be used. See Jia et al., Genome Biol. 14:R12 (2013); Haas et al. Genome Biol. 20:213 (2019).

[0063] As used herein, a “primer” refers to a chemically synthesized single-stranded oligonucleotide which is designed to anneal to a specific site on a template nucleic acid molecule. Without being limiting, a primer is used in PCR to initiate DNA synthesis. In an aspect, a primer is a DNA molecule. In an aspect, a primer is an RNA molecule. In an aspect, a primer comprises between 6 nucleotides and 70 nucleotides. In an aspect, a primer comprises between 10 nucleotides and 50 nucleotides. In an aspect, a primer comprises between 15 nucleotides and 30 nucleotides. In an aspect, a primer comprises between 18 nucleotides and 25 nucleotides. In an aspect, a primer comprises at least 6 nucleotides. In an aspect, a primer comprises at least 10 nucleotides. In an aspect, a primer comprises at least 15 nucleotides. In an aspect, a primer comprises at least 20 nucleotides. In an aspect, a primer is a forward primer. In an aspect, a primer is a reverse primer. As used herein, a “forward primer” hybridizes to the anti-sense strand of dsDNA, and a “reverse primer” hybridizes to the sense strand of dsDNA. In an aspect, a forward primer comprises DNA. In an aspect, a reverse primer comprises DNA. In an aspect, a forward primer comprises RNA. In an aspect, a reverse primer comprises RNA.

[0064] As used herein, a “target-specific primer” is a primer that is designed to hybridize (or anneal) to a single, specific target sequence at a given annealing temperature. Without being limiting, a target-specific primer is used to amplify a single target nucleotide sequence. In an aspect, a target specific primer anneals to a known target nucleotide sequence at an annealing temperature between 60°C and 75°C. In an aspect, a target specific primer anneals to a known target nucleotide sequence at an annealing temperature between 61°C and 72°C. In an aspect, a target specific primer anneals to a known target nucleotide sequence at an annealing temperature between 62°C and 70°C. In an aspect, a target specific primer anneals to a known target nucleotide sequence at an annealing temperature between 65°C and 78°C.

[0065] As used herein, when referring to two nucleic acid molecules, the terms “hybridize,” “bind,” and “anneal” are used interchangeably.

[0066] In an aspect, a primer comprises a sequence at least 80% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 85% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 90% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 95% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence at least 99% identical or complementary to a template nucleic acid molecule. In an aspect, a primer comprises a sequence 100% identical or complementary to a template nucleic acid molecule.

[0067] The polymerase chain reaction (PCR) is a molecular biology technique that allows one to generate multiple copies of a targeted region of DNA. Copies of DNA made via PCR are termed “amplicons,” “amplified product,” or “amplification product.” These amplicons contain copies of a portion of a particular target nucleic acid template strand and/or its complementary sequence, which correspond in nucleotide sequence to the template oligonucleotide sequence and/or its complementary sequence. An amplification product can further comprise sequence specific to the primers and which flanks sequence which is a portion of the target nucleic acid and/or its complement. Amplicons can then be sequenced, analyzed (e.g, gel electrophoresis), or cloned into a plasmid or vector. In an aspect, an amplicon comprises a length of between 30 nucleotides and 1000 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 750 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 500 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 250 nucleotides. In an aspect, an amplicon comprises a length of between 30 nucleotides and 100 nucleotides. In an aspect, an amplicon comprises a length of at least 20 nucleotides. In an aspect, an amplicon comprises a length of at least 25 nucleotides. In an aspect, an amplicon comprises a length of at least 30 nucleotides. In an aspect, an amplicon comprises a length of at least 50 nucleotides. In an aspect, an amplicon comprises a length of at least 75 nucleotides. In an aspect, an amplicon comprises a length of at least 100 nucleotides. In an aspect, an amplicon comprises a length of at least 125 nucleotides. In an aspect, an amplicon comprises a length of at least 150 nucleotides. In an aspect, an amplicon comprises a length of at least 175 nucleotides. In an aspect, an amplicon comprises a length of at least 200 nucleotides. In an aspect, an amplicon comprises a length of at least 250 nucleotides. In an aspect, an amplicon comprises a length of at least 300 nucleotides. In an aspect, an amplicon comprises a length of at least 400 nucleotides. In an aspect, an amplicon comprises a length of at least 500 nucleotides. In an aspect, an amplicon comprises a length of at least 600 nucleotides. In an aspect, an amplicon comprises a length of at least 700 nucleotides. In an aspect, an amplicon comprises a length of at least 800 nucleotides. In an aspect, an amplicon comprises a length of at least 900 nucleotides. In an aspect, an amplicon comprises a length of at least 1000 nucleotides. In an aspect, an amplicon comprises a length of at least 1250 nucleotides. In an aspect, an amplicon comprises a length of at least 1500 nucleotides. In an aspect, an amplicon comprises a length of at least 1750 nucleotides. In an aspect, an amplicon comprises a length of at least 2000 nucleotides. In an aspect, an amplicon is amplified from a target DNA sequence region. In an aspect, an amplicon is amplified from a template nucleic acid molecule.

[0068] In an aspect, a method comprises purifying at least one amplicon. In an aspect, a method comprises sequencing at least one amplicon. In an aspect, a method comprises re-amplification of at least one amplicon using fluorescent dideoxynucleotide triphosphates (ddNTPs). In an aspect, an amplicon is sequenced via Sanger sequencing. See Sanger and Coulson, J. Mol. Biol., 94:441- 446 (1975). In an aspect, an amplicon is sequenced via next-generation sequencing. Non-limiting examples of next-generation sequencing include single-molecule real-time sequencing (e.g., Pacific Biosciences), Ion Torrent sequencing, sequencing-by-synthesis (e.g., Illumina), sequencing by ligation (SOLiD sequencing), nanopore sequencing, and GenapSys sequencing. In another aspect, an amplicon is sequenced via Oxford Nanopore sequencing.

[0069] In an aspect, a method comprises high-throughput sequencing. In an aspect, a method comprises subjecting a plurality of amplicons to high-throughput sequencing. As used herein, “high-throughput sequencing” refers to any sequences method that is capable of sequencing multiple (e.g., tens, hundreds, thousands, millions, hundreds of millions) DNA molecules in parallel. In an aspect, Sanger sequencing is not high-throughput sequencing. In an aspect, high- throughput sequencing comprises the use of a sequencing-by-synthesis (SBS) flow cell. In an aspect, an SBS flow cell is selected from the group consisting of an Illumina SBS flow cell and a Pacific Biosciences (PacBio) SBS flow cell. In an aspect, high-throughput sequencing is performed via electrical current measurements in conjunction with an Oxford nanopore.

[0070] PCR requires a mixture comprising a targeted region of DNA to be amplified, a set of oligonucleotide primers that flank the targeted region of DNA, a thermostable DNA polymerase, and nucleotides. In general, the mixture is subjected to thermal cycling in order to amplify the targeted region of DNA. Without being limiting, thermal cycling often includes a denaturation stage to separate double-stranded DNA (dsDNA) into single strands; an annealing stage, which allows the primers to hybridize with the targeted region of DNA; and an extension stage, which allows the DNA polymerase to extend DNA from the primers, generating new dsDNA. In some protocols, the annealing and extension stages can be combined into a single stage.

[0071] In an aspect, a DNA polymerase is a thermostable DNA polymerase. As used herein, a “thermostable DNA polymerase” refers to DNA polymerases that can function at high temperatures (e.g., greater than 65°C) and can survive higher temperatures (e.g, up to about 100°C). Thermostable DNA polymerases often have maximal catalytic activity at temperatures between 70°C and 80°C. In an aspect, a thermostable DNA polymerase is selected from the group consisting of comprising Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase.

[0072] In an aspect, a DNA polymerase is a non-thermostable DNA polymerase. As used herein, a “non-thermostable DNA polymerase” refers to DNA polymerases that cannot function at high temperatures. In an aspect, a non-thermostable DNA polymerase is selected from the group consisting of phi29 DNA polymerase and Bst DNA polymerase.

[0073] In an aspect, a DNA polymerase is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase, phi29 DNA polymerase, Klenow fragment, Bst DNA polymerase, T4 DNA polymerase, Vent® DNA polymerase, LongAmp® Taq DNA polymerase, and OneTaq® DNA polymerase.

[0074] In an aspect, a DNA polymerase used in step (b) of a method provided herein is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase, phi29 DNA polymerase, Klenow fragment, Bst DNA polymerase, T4 DNA polymerase, Vent® DNA polymerase, LongAmp® Taq DNA polymerase, and OneTaq® DNA polymerase. In an aspect, a DNA polymerase used in step (c) of a method provided herein is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase [0075] As used herein, the term “blocker” refers to an oligonucleotide that is designed to selectively bind to a DNA template molecule to retard the amplification of a target sequence. In an aspect, a blocker is a DNA molecule. In an aspect, a mixture comprises a plurality of blockers. In an aspect, a blocker is an RNA molecule. In another aspect, a blocker comprises at least one continuous strand of from about 12 to about 100 nucleotides in length which strand preferably anneals to a to-be-blocked allele sequence relative to a non-blocked allele sequence, and further comprising a functional group or nucleotide sequence at its 3’ end that prevents enzymatic extension during an amplification process such as polymerase chain reaction. [0076] In an aspect, a blocker comprises between 11 and 100 nucleotides. In an aspect, a blocker comprises between 11 and 90 nucleotides. In an aspect, a blocker comprises between 11 and 80 nucleotides. In an aspect, a blocker comprises between 11 and 70 nucleotides. In an aspect, a blocker comprises between 11 and 60 nucleotides. In an aspect, a blocker comprises between 11 and 50 nucleotides. In an aspect, a blocker comprises between 11 and 40 nucleotides. In an aspect, a blocker comprises between 11 and 30 nucleotides. In an aspect, a blocker comprises between 11 and 20 nucleotides.

[0077] In an aspect, a blocker comprises at least 11 nucleotides. In an aspect, a blocker comprises at least 12 nucleotides. In an aspect, a blocker comprises at least 15 nucleotides. In an aspect, a blocker comprises at least 20 nucleotides. In an aspect, a blocker comprises at least 25 nucleotides. In an aspect, a blocker comprises at least 30 nucleotides. In an aspect, a blocker comprises at least 40 nucleotides. In an aspect, a blocker comprises at least 50 nucleotides. In an aspect, a blocker comprises at least 60 nucleotides. In an aspect, a blocker comprises at least 70 nucleotides. In an aspect, a blocker comprises at least 80 nucleotides. In an aspect, a blocker comprises at least 90 nucleotides. In an aspect, a blocker comprises at least 100 nucleotides.

[0078] In an aspect, a blocker comprises a chemical functionalization that prevents DNA polymerase extension. In an aspect, a blocker comprises a chemical functionalization that prevents DNA polymerase extension on its 3' end. In an aspect, a chemical functionalization comprises a 3- carbon spacer. In an aspect, a chemical functionalization comprises an inverted nucleotide. In an aspect, a chemical functionalization comprises a minor groove binder. In an aspect, a chemical functionalization comprises a dideoxynucleotide. In an aspect, a chemical functionalization is selected from the group consisting of a 3-carbon spacer, an inverted nucleotide, and a minor groove binder.

[0079] Typically, a blocker and a primer have partially overlapping sequences and thus they compete to bind to a given target site. The region of overlap between the blocker and primer sequences is referred to as an “overlapping subsequence.” An “overlapping subsequence” comprises a nucleotide sequence of at least 3 nucleotides of a primer sequence that is homologous with the blocker sequence.

[0080] In an aspect, a blocker and a primer comprise an overlapping sequence. In an aspect, a blocker and a forward primer comprise an overlapping sequence. In an aspect, a blocker and a reverse primer comprise an overlapping sequence. In an aspect, an overlapping sequence is positioned on the 3' end of a primer and the 5' end of a blocker. [0081] When a primer and a blocker comprise an overlapping subsequence, the primer also has a “non-overlapping subsequence,” which refers to the portion of the primer sequence that does not overlap with the blocker sequence.

[0082] In an aspect, a blocker is a wildtype-specific blocker. In an aspect, a mixture comprises a plurality of wildtype-specific blockers. In an aspect, a plurality of wildtype-specific blockers are complementary to a plurality of target DNA regions (e.g. , without being limiting, if two wildtypespecific blockers are present, one wildtype-specific blocker hybridizes (or binds) to a first locus, and the second wildtype-specific blocker hybridizes (or binds) to a second locus).

[0083] In an aspect, PCR amplification comprises one or more wildtype-specific blockers. As used herein, a “wildtype-specific blocker” refers to a blocker that is complementary to a wildtype sequence that is capable of retarding amplification of the wildtype sequence. Without being limiting, wildtype-specific blockers allow the selective amplification of non-wildtype alleles of a given locus. A wild-type-specific blocker binds to a “wildtype-specific blocker binding site” on a template nucleic acid molecule or a target DNA sequence region of interest.

[0084] In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 2 nucleotides and 30 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 3 nucleotides and 5 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 5 nucleotides and 7 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 7 nucleotides and 10 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 10 nucleotides and 15 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 15 nucleotides and 20 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 20 nucleotides and 25 nucleotides. In an aspect, a wildtype- specific blocker binding site and an IFP binding site overlap by between 25 nucleotides and 30 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 3 nucleotides and 10 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 5 nucleotides and 15 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 10 nucleotides and 20 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 15 nucleotides and 25 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 20 nucleotides and 30 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 5 nucleotides and 25 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by between 15 nucleotides and 30 nucleotides.

[0085] In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 3 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 5 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 7 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 10 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 15 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 20 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by at least 25 nucleotides.

[0086] In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 3 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 5 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 7 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 10 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 15 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 20 nucleotides. In an aspect, a wildtype-specific blocker binding site and an IFP binding site overlap by less than or equal to 25 nucleotides.

[0087] Without being limiting, an IFP can be divided into two regions when paired with a wildtype-specific blocker: a target-specific portion, which does not overlap in sequence with the wildtype-specific blocker; and an overlapping region, which does overlap in sequence with the wildtype-specific blocker. Similarly, when paired with an IFP, a wildtype-specific blocker can also be divided into two regions: a blocker-unique sequence, which does not overlap in sequences with the IFP; and the overlapping region, which does overlap in sequence with the IFP.

[0088] In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -1 kcal/mol and -3 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -2 kcal/mol and -4 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -3 kcal/mol and -5 kcal/mol. In an aspect, an overlapping region between a wildtype- specific blocker and an IFP comprises a standard free energy of binding between -4 kcal/mol and -6 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -5 kcal/mol and -7 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -6 kcal/mol and -8 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -7 kcal/mol and -9 kcal/mol. In an aspect, an overlapping region between a wildtype- specific blocker and an IFP comprises a standard free energy of binding between -8 kcal/mol and -10 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -2 kcal/mol and -8 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -2 kcal/mol and -10 kcal/mol. In an aspect, an overlapping region between a wildtype-specific blocker and an IFP comprises a standard free energy of binding between -5 kcal/mol and -10 kcal/mol.

[0089] In an aspect, the target-specific portion of an IFP that does not overlap with a wildtype- specific blocker comprises a standard free energy of binding between -1 kcal/mol and -5 kcal/mol. In an aspect, the target-specific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -2 kcal/mol and -6 kcal/mol. In an aspect, the target-specific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -3 kcal/mol and -7 kcal/mol. In an aspect, the target-specific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -4 kcal/mol and -8 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -5 kcal/mol and -9 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -6 kcal/mol and -10 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -7 kcal/mol and -11 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -8 kcal/mol and -12 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -9 kcal/mol and -13 kcal/mol. In an aspect, the targetspecific portion of an IFP that does not overlap with a wildtype-specific blocker comprises a standard free energy of binding between -1 kcal/mol and -13 kcal/mol. [0090] In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -1 kcal/mol and -6 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -2 kcal/mol and -7 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -3 kcal/mol and -8 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -4 kcal/mol and -9 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -5 kcal/mol and -10 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -6 kcal/mol and -11 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -7 kcal/mol and -12 kcal/mol. In an aspect, the blocker-unique sequence of a wildtypespecific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -8 kcal/mol and -13 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -9 kcal/mol and -14 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -10 kcal/mol and -15 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -11 kcal/mol and -16 kcal/mol. In an aspect, the blockerunique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -1 kcal/mol and -16 kcal/mol. In an aspect, the blocker-unique sequence of a wildtype-specific blocker that does not overlap with an IFP sequence comprises a standard free energy of binding between -7 kcal/mol and -16 kcal/mol.

[0091] Unless described otherwise, the standard free energy of binding is calculated based on an annealing temperature of 60°C, double-stranded DNA, and aNa ⁺ concentration of 0.18 M.

[0092] In an aspect, an IFP, OFP, or an MFP comprises a standard free energy of binding of between -11.5 kcal/mol and -12.5 kcal/mol in a standard PCR buffer.

[0093] In an aspect, a wildtype-specific blocker comprises a terminator to prevent 3' to 5' DNA polymerase exonuclease activity. In an aspect, a terminator is selected from the group consisting of a three-carbon (C3) spacer and DXXDM, where D is a match between the blocker sequence and the template nucleic acid molecule sequence or target DNA region, M is a C3 spacer, and X is mismatch between the blocker sequence and the template nucleic acid sequence or target DNA region. Additional terminators known in the art are also suitable for use. A non-limiting example of an additional terminator is a dideoxynucleotide. In an aspect, a wildtype-specific blocker comprises a terminator comprising a DNA overhang comprising four nucleotides.

[0094] As used herein, the term “nucleic acid” or “nucleic acid sequence” refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or an analog thereof. A nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one strand nucleic acid of a denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, a template nucleic acid is DNA. In another aspect, a template is RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA. In an aspect, a nucleic acid molecule provided herein is a DNA molecule. In an aspect, a nucleic acid molecule provided herein is an RNA molecule. In an aspect, a nucleic acid molecule provided herein is a cDNA molecule.

[0095] As used herein, “DNA” refers to deoxyribonucleic acid. DNA can be either singlestranded or double-stranded. In an aspect, DNA comprises complementary DNA (cDNA). DNA typically comprises four nucleotides: cytosine (C), guanine (G), adenine (A), and thymine (T). In an aspect, the sequence of a DNA molecule provided herein comprises one or more degenerate nucleotides. As used herein, a “degenerate nucleotide” refers to a nucleotide that can perform the same function or yield the same output as a structurally different nucleotide. Non-limiting examples of degenerate nucleotides include a C, G, or T nucleotide (B); an A, G, or T nucleotide (D); an A, C, or T nucleotide (H); a G or T nucleotide (K); an A or C nucleotide (M); any nucleotide (N); an A or G nucleotide (R); a G or C nucleotide (S); an A, C, or G nucleotide (V); an A or T nucleotide (W), and a C or T nucleotide (Y).

[0096] As used herein, “specific” or “specifically” when used in the context of a primer specific for a target nucleic acid refers to a level of complementarity between the primer and the target such that there exists an annealing temperature at which the primer will anneal to and mediate amplification of the target nucleic acid and will not anneal to or mediate amplification of nontarget sequences present in a sample.

[0097] In an aspect, this application provides a method of preparing a nucleic acid template into a DNA library for sequencing, the method comprising the steps of: (a) mixing a nucleic acid template, a Ligation Tail Adapter (LTA) molecule, a DNA ligase, and reagents for DNA ligase activity to form a first mixture; where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand which two strands form a Double-Stranded End (DSE) and a DNA Tail (DT) region; (b) subjecting the first mixture to a suitable temperature to allow for DNA ligation to form a ligation product mixture; (c) introducing to the ligation product mixture from step (b) a targetspecific Outer Forward Primer (OFP) and a Splint, a DNA polymerase, and reagents for DNA polymerase activity to form a second mixture; where the OFP comprises a nucleic acid sequence that can specifically anneal to a portion of a target DNA sequence region of interest, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region or portion thereof; (d) subjecting the second mixture to a suitable temperature to allow for DNA polymerase extension to form a DNA polymerase extension product mixture; (e) introducing to the DNA polymerase extension product mixture from step (d) a target-specific Inner Forward Primer (IFP), a Universal Primer (UP), a thermostable DNA polymerase, and reagents for DNA polymerase activity to form a third mixture, where the IFP comprises a nucleic acid sequence that can specifically anneal to a portion of the target DNA sequence region of interest; and where the UP comprises a sequence which can specifically anneal to the 5' end subsequence, or portion thereof, of the Splint; and (1) subjecting the third mixture to temperature cycles to allow for polymerase chain reaction (PCR) amplification. [0098] In an aspect, the DNA polymerase extension of step (d), the PCR amplification of step (f), or both, are multiplexed.

[0099] In an aspect, a ligation produce is purified after step (a) of a method provided herein. In an aspect, purification comprises column purification. In an aspect, purification comprises beads purification. In an aspect, purification comprises the use of phenol and/or chloroform. In an aspect, purification comprises diluting the ligation product between 10-fold and 10,000-fold. In an aspect, purification comprises a method selected from the group consisting of column purification, beads purification, diluting the ligation product between 10-fold and 10,000-fold, and any combination thereof.

[0100] In an aspect, purification comprises diluting the ligation product at least 20-fold. In an aspect, purification comprises diluting the ligation product at least 50-fold. In an aspect, purification comprises diluting the ligation product at least 100-fold. In an aspect, purification comprises diluting the ligation product at least 200-fold. In an aspect, purification comprises diluting the ligation product at least 500-fold. In an aspect, purification comprises diluting the ligation product at least 1000-fold. In an aspect, purification comprises diluting the ligation product at least 2000-fold. In an aspect, purification comprises diluting the ligation product at least 5000- fold. In an aspect, purification comprises diluting the ligation product at least 7500-fold. In an aspect, purification comprises diluting the ligation product at least 9000-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 20-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 50-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 100-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 200-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 500-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 1000-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 2000-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 5000-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 7500-fold. In an aspect, purification comprises diluting the ligation product less than or equal to 9000-fold.

[0101] In an aspect, this application provides a method for preparing a nucleic acid for sequencing, the method comprising: (i) ligating a Ligation Tail Adapter (LTA) molecule to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand, and where the LTA- top strand and the LTA-bottom strand form a Double-Stranded End (DSE) and a DNA Tail (DT) region to produce a ligation product; (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a Splint, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region to produce an amplification product; and (iii) amplifying an amplification product using a second target-specific primer that specifically anneals to the amplification product and a Universal Primer (UP) comprising a sequence which can specifically anneal to the 5' end subsequence, or a portion thereof, of the Splint, where the second target-specific primer is nested relative to the first target-specific primer.

[0102] As used herein, a “Universal Primer” or “UP” refers to a primer that is complementary to nucleotide sequences that are common in a particular set of DNA molecules. Without being limiting, universal primers are able to bind to a wide variety of DNA templates (e.g, they are not specific to a single locus).

[0103] In an aspect, a UP comprises between 10 nucleotides and 70 nucleotides. In an aspect, a UP comprises between 10 nucleotides and 15 nucleotides. In an aspect, a UP comprises between 15 nucleotides and 20 nucleotides. In an aspect, a UP comprises between 20 nucleotides and 25 nucleotides. In an aspect, a UP comprises between 25 nucleotides and 30 nucleotides. In an aspect, a UP comprises between 30 nucleotides and 35 nucleotides. In an aspect, a UP comprises between 35 nucleotides and 40 nucleotides. In an aspect, a UP comprises between 40 nucleotides and 45 nucleotides. In an aspect, a UP comprises between 45 nucleotides and 50 nucleotides. In an aspect, a UP comprises between 50 nucleotides and 55 nucleotides. In an aspect, a UP comprises between 55 nucleotides and 60 nucleotides. In an aspect, a UP comprises between 60 nucleotides and 65 nucleotides. In an aspect, a UP comprises between 65 nucleotides and 70 nucleotides. In an aspect, a UP comprises between 10 nucleotides and 20 nucleotides. In an aspect, a UP comprises between 15 nucleotides and 25 nucleotides. In an aspect, a UP comprises between 20 nucleotides and 30 nucleotides. In an aspect, a UP comprises between 25 nucleotides and 35 nucleotides. In an aspect, a UP comprises between 35 nucleotides and 45 nucleotides. In an aspect, a UP comprises between 10 nucleotides and 25 nucleotides. In an aspect, a UP comprises between 15 nucleotides and 50 nucleotides. In an aspect, a UP comprises between 20 nucleotides and 55 nucleotides. In an aspect, a UP comprises between 35 nucleotides and 60 nucleotides. In an aspect, a UP comprises between 50 nucleotides and 70 nucleotides.

[0104] In an aspect, a UP comprises at least 12 nucleotides. In an aspect, a UP comprises at least 15 nucleotides. In an aspect, a UP comprises at least 20 nucleotides. In an aspect, a UP comprises at least 25 nucleotides. In an aspect, a UP comprises at least 30 nucleotides. In an aspect, a UP comprises at least 35 nucleotides. In an aspect, a UP comprises at least 40 nucleotides. In an aspect, a UP comprises at least 45 nucleotides. In an aspect, a UP comprises at least 50 nucleotides. In an aspect, a UP comprises at least 55 nucleotides. In an aspect, a UP comprises at least 60 nucleotides.

[0105] In an aspect, a UP comprises less than or equal to 12 nucleotides. In an aspect, a UP comprises less than or equal to 15 nucleotides. In an aspect, a UP comprises less than or equal to 20 nucleotides. In an aspect, a UP comprises less than or equal to 25 nucleotides. In an aspect, a UP comprises less than or equal to 30 nucleotides. In an aspect, a UP comprises less than or equal to 35 nucleotides. In an aspect, a UP comprises less than or equal to 40 nucleotides. In an aspect, a UP comprises less than or equal to 45 nucleotides. In an aspect, a UP comprises less than or equal to 50 nucleotides. In an aspect, a UP comprises less than or equal to 55 nucleotides. In an aspect, a UP comprises less than or equal to 60 nucleotides.

[0106] In an aspect, a UP contains no sequence that is complementary to any 5 -nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 6-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 7-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 8- nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 9-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 10-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 11 -nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 12-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 13-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 14-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 15-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 16-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 17-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 18-nucleotide (nt) or longer continuous subsequence of an LTA. In an aspect, a UP contains no sequence that is complementary to any 19-nucleotide (nt) or longer continuous subsequence of an LTA.

[0107] In an aspect, a primer is an Inner Forward Primer (IFP). In an aspect, an IFP binds (e.g. , hybridizes) to an IFP binding site on a template nucleic acid molecule. In an aspect, a primer is an Outer Forward Primer (OFP). In an aspect, an OFP binds (e.g , hybridizes) to an OFP binding site on a template nucleic acid molecule. In an aspect, a primer is a Middle Forward Primer (MFP). In an aspect, an MFP binds (e.g., hybridizes) to an MFP binding site on a template nucleic acid molecule. For a given template nucleic acid molecule, gene or target sequence, an IFP binding site is positioned, at least partially, 3’ to an OFP binding site. For a given gene or target sequence, an MFP binding site partially overlaps an OFP binding site, an IFP binding site, or both. In an aspect, an IFP binding site, an OFP binding site, or an MFP binding site is on the positive strand of a DNA or cDNA molecule. In an aspect, an IFP binding site, an OFP binding site, or an MFP binding site is on the negative strand of a DNA or cDNA molecule.

[0108] In an aspect, a method comprises a plurality of OFPs. In an aspect, a method comprises a plurality of IFPs. In an aspect, a method comprises a plurality of MFPs. As used herein, a “plurality” refers to 2 or more. In an aspect, a plurality comprises 3 or more. In an aspect, a plurality comprises 4 or more. In an aspect, a plurality comprises 5 or more. In an aspect, a plurality comprises 10 or more. In an aspect, a plurality comprises 15 or more. In an aspect, a plurality comprises 20 or more. In an aspect, a plurality comprises 30 or more. [0109] In an aspect, an IFP and an OFP comprise overlapping sequences. In an aspect, an IFP and an OFP do not comprise overlapping sequences. In an aspect, an IFP and an OFP have a partially or completely overlapping binding site on a target DNA sequence region of interest.

[0110] In an aspect, an OFP comprises between 10 nucleotides and 100 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 90 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 80 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 70 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 60 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 50 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 40 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 30 nucleotides. In an aspect, an OFP comprises between 10 nucleotides and 20 nucleotides. In an aspect, an OFP comprises between 15 nucleotides and 90 nucleotides. In an aspect, an OFP comprises between 20 nucleotides and 80 nucleotides. In an aspect, an OFP comprises between 25 nucleotides and 70 nucleotides. In an aspect, an OFP comprises between 30 nucleotides and 60 nucleotides. In an aspect, an OFP comprises between 35 nucleotides and 50 nucleotides. In an aspect, an OFP comprises between 15 nucleotides and 50 nucleotides. In an aspect, an OFP comprises between 20 nucleotides and 40 nucleotides. In an aspect, an OFP comprises between 25 nucleotides and 30 nucleotides. In an aspect, an OFP comprises between 20 nucleotides and 30 nucleotides.

[OHl] In an aspect, an OFP comprises a 5' overhang which does not bind to the reverse strand of the nucleic acid template. In an aspect, a 5' overhang comprises between 1 nucleotide and 100 nucleotides. In an aspect, a 5' overhang comprises between 2 nucleotides and 95 nucleotides. In an aspect, a 5' overhang comprises between 3 nucleotides and 90 nucleotides. In an aspect, a 5' overhang comprises between 4 nucleotides and 80 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 70 nucleotides. In an aspect, a 5' overhang comprises between 6 nucleotides and 60 nucleotides. In an aspect, a 5' overhang comprises between 7 nucleotides and 50 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 90 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 80 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 70 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 60 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 50 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 40 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 30 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 20 nucleotides. In an aspect, a 5' overhang comprises between 5 nucleotides and 10 nucleotides. In an aspect, a 5' overhang comprises between 10 nucleotides and 20 nucleotides. In an aspect, a 5' overhang comprises between 15 nucleotides and 25 nucleotides. In an aspect, a 5' overhang comprises between 20 nucleotides and 30 nucleotides. In an aspect, a 5' overhang comprises between 25 nucleotides and 35 nucleotides. In an aspect, a 5' overhang comprises between 30 nucleotides and 40 nucleotides. In an aspect, a 5' overhang comprises between 35 nucleotides and 45 nucleotides. In an aspect, a 5' overhang comprises between 40 nucleotides and 50 nucleotides. In an aspect, a 5' overhang comprises between 45 nucleotides and 55 nucleotides. In an aspect, a 5' overhang comprises between 50 nucleotides and 60 nucleotides. In an aspect, a 5' overhang comprises between 55 nucleotides and 65 nucleotides. In an aspect, a 5' overhang comprises between 60 nucleotides and 70 nucleotides. In an aspect, a 5' overhang comprises between 65 nucleotides and 75 nucleotides. In an aspect, a 5' overhang comprises between 70 nucleotides and 80 nucleotides. In an aspect, a 5' overhang comprises between 75 nucleotides and 85 nucleotides. In an aspect, a 5' overhang comprises between 10 nucleotides and 80 nucleotides. In an aspect, a 5' overhang comprises between 15 nucleotides and 70 nucleotides. In an aspect, a 5' overhang comprises between 20 nucleotides and 60 nucleotides. In an aspect, a 5' overhang comprises between 25 nucleotides and 50 nucleotides.

[0112] In an aspect, an OFP anneals to a template nucleic acid at a temperature between 55°C and 72°C. In an aspect, an IFP anneals to a template nucleic acid at a temperature between 55°C and 72°C.

[0113] In an aspect, an IFP comprises between 10 nucleotides and 70 nucleotides. In an aspect, an IFP comprises between 10 nucleotides and 15 nucleotides. In an aspect, an IFP comprises between 15 nucleotides and 20 nucleotides. In an aspect, an IFP comprises between 20 nucleotides and 25 nucleotides. In an aspect, an IFP comprises between 25 nucleotides and 30 nucleotides. In an aspect, an IFP comprises between 30 nucleotides and 35 nucleotides. In an aspect, an IFP comprises between 35 nucleotides and 40 nucleotides. In an aspect, an IFP comprises between 40 nucleotides and 45 nucleotides. In an aspect, an IFP comprises between 45 nucleotides and 50 nucleotides. In an aspect, an IFP comprises between 50 nucleotides and 55 nucleotides. In an aspect, an IFP comprises between 55 nucleotides and 60 nucleotides. In an aspect, an IFP comprises between 60 nucleotides and 65 nucleotides. In an aspect, an IFP comprises between 65 nucleotides and 70 nucleotides. In an aspect, an IFP comprises between 10 nucleotides and 20 nucleotides. In an aspect, an IFP comprises between 15 nucleotides and 25 nucleotides. In an aspect, an IFP comprises between 20 nucleotides and 30 nucleotides. In an aspect, an IFP comprises between 25 nucleotides and 35 nucleotides. In an aspect, an IFP comprises between 35 nucleotides and 45 nucleotides. In an aspect, an IFP comprises between 10 nucleotides and 25 nucleotides. In an aspect, an IFP comprises between 15 nucleotides and 50 nucleotides. In an aspect, an IFP comprises between 20 nucleotides and 55 nucleotides. In an aspect, an IFP comprises between 35 nucleotides and 60 nucleotides. In an aspect, an IFP comprises between 50 nucleotides and 70 nucleotides.

[0114] In an aspect, an IFP comprises at least 12 nucleotides. In an aspect, an IFP comprises at least 15 nucleotides. In an aspect, an IFP comprises at least 20 nucleotides. In an aspect, an IFP comprises at least 25 nucleotides. In an aspect, an IFP comprises at least 30 nucleotides. In an aspect, an IFP comprises at least 35 nucleotides. In an aspect, an IFP comprises at least 40 nucleotides. In an aspect, an IFP comprises at least 45 nucleotides. In an aspect, an IFP comprises at least 50 nucleotides. In an aspect, an IFP comprises at least 55 nucleotides. In an aspect, an IFP comprises at least 60 nucleotides.

[0115] In an aspect, an IFP comprises less than or equal to 12 nucleotides. In an aspect, an IFP comprises less than or equal to 15 nucleotides. In an aspect, an IFP comprises less than or equal to 20 nucleotides. In an aspect, an IFP comprises less than or equal to 25 nucleotides. In an aspect, an IFP comprises less than or equal to 30 nucleotides. In an aspect, an IFP comprises less than or equal to 35 nucleotides. In an aspect, an IFP comprises less than or equal to 40 nucleotides. In an aspect, an IFP comprises less than or equal to 45 nucleotides. In an aspect, an IFP comprises less than or equal to 50 nucleotides. In an aspect, an IFP comprises less than or equal to 55 nucleotides. In an aspect, an IFP comprises less than or equal to 60 nucleotides.

[0116] In an aspect, an IFP binds atemplate nucleic acid 5' relative to an OFP, and the IFP and OFP do not overlap when bound to the template nucleic acid. In an aspect, an IFP binding site and an OFP binding site do not overlap.

[0117] In an aspect, an IFP binding site is separated from a breakpoint of a known template region by a gap distance comprising between 5 nucleotides and 30 nucleotides. In an aspect, a gap distance comprises at least 7 nucleotides. In an aspect, a gap distance comprises at least 10 nucleotides. In an aspect, a gap distance comprises at least 15 nucleotides. In an aspect, a gap distance comprises at least 20 nucleotides. In an aspect, a gap distance comprises at least 25 nucleotides. In an aspect, a gap distance comprises less than or equal to 7 nucleotides. In an aspect, a gap distance comprises less than or equal to 10 nucleotides. In an aspect, a gap distance comprises less than or equal to 15 nucleotides. In an aspect, a gap distance comprises less than or equal to 20 nucleotides. In an aspect, a gap distance comprises less than or equal to 25 nucleotides. In an aspect, a gap distance comprises between 5 nucleotides and 7 nucleotides. In an aspect, a gap distance comprises between 7 nucleotides and 10 nucleotides. In an aspect, a gap distance comprises between 10 nucleotides and 15 nucleotides. In an aspect, a gap distance comprises between 15 nucleotides and 20 nucleotides. In an aspect, a gap distance comprises between 20 nucleotides and 25 nucleotides. In an aspect, a gap distance comprises between 25 nucleotides and 30 nucleotides. In an aspect, a gap distance comprises between 5 nucleotides and 15 nucleotides. In an aspect, a gap distance comprises between 10 nucleotides and 20 nucleotides. In an aspect, a gap distance comprises between 15 nucleotides and 25 nucleotides. In an aspect, a gap distance comprises between 20 nucleotides and 30 nucleotides.

[0118] When two primer binding sites are positioned such that there are 0 nucleotides between them, and the two primer binding sites do not overlap, the primer binding sites are considered to be “adjacent.” In an aspect, an OFP binding site and an IFP binding site are adjacent. In an aspect, an OFP binding site and an IFP binding site are adjacent.

[0119] In an aspect, an IFP binding site and an OFP binding site overlap. When binding sites “overlap” it means that the same sequence in a nucleic acid molecule (e.g., a nucleic acid template) is complementary to both an IFP (in part) and an OFP (in part).

[0120] As used herein, an “IFP binding site” refers to the location in a nucleic acid molecule to which an IFP is capable of binding. Typically, an IFP binding site will be completely, or partially, complementary to the IFP. Similarly, an “OFP binding site” refers to the location in a nucleic acid molecule to which an OFP is capable of binding. Typically, an OFP binding site will be completely, or partially, complementary to the OFP. When an IFP binding site and an OFP binding site overlap, the overlapping sequence is termed an “overlapping region.” Any binding site provided herein (e.g., an IFP binding site, an OFP binding site, a MFP binding site, a blocker binding site) can be positioned within a template nucleic acid molecule or a target DNA sequence region of interest.

[0121] In an aspect, an overlapping region comprises between 1 nucleotide and 40 nucleotides. In an aspect, an overlapping region comprises between 3 nucleotides and 5 nucleotides. In an aspect, an overlapping region comprises between 5 nucleotides and 7 nucleotides. In an aspect, an overlapping region comprises between 7 nucleotides and 10 nucleotides. In an aspect, an overlapping region comprises between 10 nucleotides and 15 nucleotides. In an aspect, an overlapping region comprises between 15 nucleotides and 20 nucleotides. In an aspect, an overlapping region comprises between 20 nucleotides and 25 nucleotides. In an aspect, an overlapping region comprises between 25 nucleotides and 30 nucleotides. In an aspect, an overlapping region comprises between 30 nucleotides and 35 nucleotides. In an aspect, an overlapping region comprises between 35 nucleotides and 40 nucleotides. In an aspect, an overlapping region comprises between 3 nucleotides and 10 nucleotides. In an aspect, an overlapping region comprises between 5 nucleotides and 15 nucleotides. In an aspect, an overlapping region comprises between 10 nucleotides and 20 nucleotides. In an aspect, an overlapping region comprises between 15 nucleotides and 25 nucleotides. In an aspect, an overlapping region comprises between 20 nucleotides and 30 nucleotides. In an aspect, an overlapping region comprises between 25 nucleotides and 35 nucleotides. In an aspect, an overlapping region comprises between 5 nucleotides and 25 nucleotides. In an aspect, an overlapping region comprises between 15 nucleotides and 40 nucleotides. In an aspect, an overlapping region comprises between 20 nucleotides and 40 nucleotides.

[0122] In an aspect, an overlapping region comprises at least 3 nucleotides. In an aspect, an overlapping region comprises at least 5 nucleotides. In an aspect, an overlapping region comprises at least 7 nucleotides. In an aspect, an overlapping region comprises at least 10 nucleotides. In an aspect, an overlapping region comprises at least 15 nucleotides. In an aspect, an overlapping region comprises at least 20 nucleotides. In an aspect, an overlapping region comprises at least 25 nucleotides. In an aspect, an overlapping region comprises at least 30 nucleotides. In an aspect, an overlapping region comprises at least 35 nucleotides.

[0123] In an aspect, an overlapping region comprises less than or equal to 3 nucleotides. In an aspect, an overlapping region comprises less than or equal to 5 nucleotides. In an aspect, an overlapping region comprises less than or equal to 7 nucleotides. In an aspect, an overlapping region comprises less than or equal to 10 nucleotides. In an aspect, an overlapping region comprises less than or equal to 15 nucleotides. In an aspect, an overlapping region comprises less than or equal to 20 nucleotides. In an aspect, an overlapping region comprises less than or equal to 25 nucleotides. In an aspect, an overlapping region comprises less than or equal to 30 nucleotides. In an aspect, an overlapping region comprises less than or equal to 35 nucleotides.

[0124] When two primer binding sites do not overlap and are not adjacent, they are considered to have a “gap” between them. In an aspect, there is a gap between an OFP binding site and an IFP binding site.

[0125] In an aspect, a gap comprises between 1 nucleotide and 50 nucleotides. In an aspect, a gap comprises between 3 nucleotides and 5 nucleotides. In an aspect, a gap comprises between 5 nucleotides and 7 nucleotides. In an aspect, a gap comprises between 7 nucleotides and 10 nucleotides. In an aspect, a gap comprises between 10 nucleotides and 15 nucleotides. In an aspect, a gap comprises between 15 nucleotides and 20 nucleotides. In an aspect, a gap comprises between 20 nucleotides and 25 nucleotides. In an aspect, a gap comprises between 25 nucleotides and 30 nucleotides. In an aspect, a gap comprises between 30 nucleotides and 35 nucleotides. In an aspect, a gap comprises between 35 nucleotides and 40 nucleotides. In an aspect, a gap comprises between 3 nucleotides and 10 nucleotides. In an aspect, a gap comprises between 5 nucleotides and 15 nucleotides. In an aspect, a gap comprises between 10 nucleotides and 20 nucleotides. In an aspect, a gap comprises between 15 nucleotides and 25 nucleotides. In an aspect, a gap comprises between 20 nucleotides and 30 nucleotides. In an aspect, a gap comprises between 25 nucleotides and 35 nucleotides. In an aspect, a gap comprises between 5 nucleotides and 25 nucleotides. In an aspect, a gap comprises between 15 nucleotides and 40 nucleotides. In an aspect, a gap comprises between 20 nucleotides and 40 nucleotides.

[0126] In an aspect, a gap comprises at least 3 nucleotides. In an aspect, a gap comprises at least 5 nucleotides. In an aspect, a gap comprises at least 7 nucleotides. In an aspect, a gap comprises at least 10 nucleotides. In an aspect, a gap comprises at least 15 nucleotides. In an aspect, a gap comprises at least 20 nucleotides. In an aspect, a gap comprises at least 25 nucleotides. In an aspect, a gap comprises at least 30 nucleotides. In an aspect, a gap comprises at least 35 nucleotides. [0127] In an aspect, a gap comprises less than or equal to 3 nucleotides. In an aspect, a gap comprises less than or equal to 5 nucleotides. In an aspect, a gap comprises less than or equal to 7 nucleotides. In an aspect, a gap comprises less than or equal to 10 nucleotides. In an aspect, a gap comprises less than or equal to 15 nucleotides. In an aspect, a gap comprises less than or equal to 20 nucleotides. In an aspect, a gap comprises less than or equal to 25 nucleotides. In an aspect, a gap comprises less than or equal to 30 nucleotides. In an aspect, a gap comprises less than or equal to 35 nucleotides.

[0128] In an aspect, use of an OFP and an IFP provide a nested polymerase chain reaction (PCR) that further comprises a middle PCR to improve the specificity and on-target rate. In an aspect, a middle PCR comprises using an MFP that binds to an MFP binding site on at least one template nucleic acid molecule, where the MFP binding site partially overlaps with the OFP binding site, the IFP binding site, or both. In an aspect, a middle PCR comprises using an MFP that comprises a 5' region starting from the second nucleotide of the OFP 5' region to the second nucleotide of the OFP 3' region, and the MFP comprises a 3' region starting from the second nucleotide of the IFP 5' region to the second nucleotide of the IFP 3' region.

[0129] In an aspect, an MFP comprises between 10 nucleotides and 70 nucleotides. In an aspect, an MFP comprises between 10 nucleotides and 15 nucleotides. In an aspect, an MFP comprises between 15 nucleotides and 20 nucleotides. In an aspect, an MFP comprises between 20 nucleotides and 25 nucleotides. In an aspect, an MFP comprises between 25 nucleotides and 30 nucleotides. In an aspect, an MFP comprises between 30 nucleotides and 35 nucleotides. In an aspect, an MFP comprises between 35 nucleotides and 40 nucleotides. In an aspect, an MFP comprises between 40 nucleotides and 45 nucleotides. In an aspect, an MFP comprises between 45 nucleotides and 50 nucleotides. In an aspect, an MFP comprises between 50 nucleotides and 55 nucleotides. In an aspect, an MFP comprises between 55 nucleotides and 60 nucleotides. In an aspect, an MFP comprises between 60 nucleotides and 65 nucleotides. In an aspect, an MFP comprises between 65 nucleotides and 70 nucleotides. In an aspect, an MFP comprises between 10 nucleotides and 20 nucleotides. In an aspect, an MFP comprises between 15 nucleotides and 25 nucleotides. In an aspect, an MFP comprises between 20 nucleotides and 30 nucleotides. In an aspect, an MFP comprises between 25 nucleotides and 35 nucleotides. In an aspect, an MFP comprises between 35 nucleotides and 45 nucleotides. In an aspect, an MFP comprises between 10 nucleotides and 25 nucleotides. In an aspect, an MFP comprises between 15 nucleotides and 50 nucleotides. In an aspect, an MFP comprises between 20 nucleotides and 55 nucleotides. In an aspect, an MFP comprises between 35 nucleotides and 60 nucleotides. In an aspect, an MFP comprises between 50 nucleotides and 70 nucleotides.

[0130] In an aspect, an MFP comprises at least 12 nucleotides. In an aspect, an MFP comprises at least 15 nucleotides. In an aspect, an MFP comprises at least 20 nucleotides. In an aspect, an MFP comprises at least 25 nucleotides. In an aspect, an MFP comprises at least 30 nucleotides. In an aspect, an MFP comprises at least 35 nucleotides. In an aspect, an MFP comprises at least 40 nucleotides. In an aspect, an MFP comprises at least 45 nucleotides. In an aspect, an MFP comprises at least 50 nucleotides. In an aspect, an MFP comprises at least 55 nucleotides. In an aspect, an MFP comprises at least 60 nucleotides.

[0131] In an aspect, an MFP comprises less than or equal to 12 nucleotides. In an aspect, an MFP comprises less than or equal to 15 nucleotides. In an aspect, an MFP comprises less than or equal to 20 nucleotides. In an aspect, an MFP comprises less than or equal to 25 nucleotides. In an aspect, an MFP comprises less than or equal to 30 nucleotides. In an aspect, an MFP comprises less than or equal to 35 nucleotides. In an aspect, an MFP comprises less than or equal to 40 nucleotides. In an aspect, an MFP comprises less than or equal to 45 nucleotides. In an aspect, an MFP comprises less than or equal to 50 nucleotides. In an aspect, an MFP comprises less than or equal to 55 nucleotides. In an aspect, an MFP comprises less than or equal to 60 nucleotides.

[0132] In an aspect, an IFP comprises a 5'-end single-stranded nucleic acid sequence. In an aspect, an IFP comprises a 5 '-end single-stranded nucleic acid sequence which does not bind to a nucleic acid template. In an aspect, a 5'-end single-stranded nucleic acid sequence comprises a sequencing adapter. In an aspect, a UP comprises a sequencing adapter. In an aspect, a sequencing adapter is an Illumina sequencing adapter. In an aspect, a sequencing adapter is a nanopore sequencing adapter. In an aspect, a sequencing adapter is an Ion Torrent sequencing adapter. As used herein, a “sequencing adapter” refers to a sequencing platform-specific sequence used for fragment recognition by a sequencing instrument. In an aspect, a sequencing adapter is a full-length sequencing adapter. In an aspect, a sequencing adapter is a partial sequencing adapter. In an aspect, a UP comprises a partial sequencing adapter which is amplified with a sequencing index primer. [0133] As used herein, a “forward primer” hybridizes to the anti-sense strand of a dsDNA molecule, and a “reverse primer” hybridizes to the sense strand of the dsDNA molecule.

[0134] As used herein, a “nucleic acid template” refers to a nucleic acid molecule that is to be amplified in whole or in part. In an aspect, a nucleic acid template comprises a target DNA sequence region of interest. In an aspect, a nucleic acid template comprises DNA. In an aspect, a nucleic acid template comprises cDNA. As is appreciated in the art, cDNA molecules can be generated through the reverse transcription of an RNA sample (particularly a messenger RNA sample). In an aspect, a nucleic acid template comprises double-stranded DNA. In an aspect, a nucleic acid template comprises RNA. In an aspect, a nucleic acid template comprises an amplicon DNA molecule generated by a DNA polymerase. In an aspect, a nucleic acid template is from a physically, chemically, or enzymatically treated product of a biological DNA or RNA sample. In an aspect, a nucleic acid template is from a product of a fragmentation process. Non-limiting examples of a fragmentation process include ultrasonication and enzymatic fragmentation. In an aspect, a nucleic acid template undergoes an end-repair process prior to the initiation of any method provided herein (e.g., before step (a) of a method). In an aspect, an end-repair process is performed using a T4 DNA ligase enzyme. In an aspect, a nucleic acid template is ligated using a blunt TA ligase enzyme.

[0135] In an aspect, a nucleic acid template is a biological DNA derived from a sample of cells from biofluids such as blood, urine, saliva, feces, cerebrospinal fluid, interstitial fluid, and synovial fluid, or from a tissue such as a biopsy tissue or a surgically resected tissue.

[0136] In an aspect, a nucleic acid template is a eukaryotic DNA molecule. In an aspect, a nucleic acid template is a prokaryotic DNA molecule. In an aspect, a nucleic acid template is a viral DNA molecule. In an aspect, a nucleic acid template is a viroid DNA molecule.

[0137] In an aspect, a nucleic acid template is selected from an animal nucleic acid molecule, a plant nucleic acid molecule, a fungal nucleic acid molecule, and a protozoan nucleic acid molecule. In an aspect, a nucleic acid template is selected from a bacterial nucleic acid molecule and an archaea nucleic acid molecule. In an aspect, a nucleic acid template molecule is selected from the group consisting of an Adenoviridae nucleic acid molecule, a Herpesviridae nucleic acid molecule, a Poxviridae nucleic acid molecule, a Papillomaviridae nucleic acid molecule, a Parvoviridae nucleic acid molecule, a Reoviridae nucleic acid molecule, a Coronaviridae nucleic acid molecule, a Picomaviridae nucleic acid molecule, a Togaviridae nucleic acid molecule, an Orthomyxoviridae nucleic acid molecule, a Rhabdoviridae nucleic acid molecule, a Retroviridae nucleic acid molecule, a Hepadnaviridae nucleic acid molecule, a Baculoviridae nucleic acid molecule, a Geminiviridae nucleic acid molecule, a Flaviviridae nucleic acid molecule, a Filoviridae nucleic acid molecule, a Paramyxoviridae nucleic acid molecule, and a Pneumoviridae nucleic acid molecule. In an aspect, a virus nucleic acid molecule is selected from the group consisting of a human orthopnemovirus (HRSV) nucleic acid molecule, an influenza virus nucleic acid molecule, a human immunodeficiency virus (HIV) nucleic acid molecule, a hepatitis B virus (HBV) nucleic acid molecule, and a human papillomavirus (HPV) nucleic acid molecule.

[0138] In an aspect, a nucleic acid template molecule is selected from the group consisting of a Pospiviroidae nucleic acid molecule, and a Avsunviroidae nucleic acid molecule.

[0139] In an aspect, a nucleic acid template is a human DNA molecule. In an aspect, a nucleic acid template is an animal DNA molecule. In an aspect, a nucleic acid template is a rodent DNA molecule. In an aspect, a nucleic acid template is a plant DNA molecule. In an aspect, a nucleic acid template is a fungal DNA molecule. In an aspect, a nucleic acid template is an environmental specimen DNA molecule.

[0140] As used herein, a “target DNA sequence region of interest” refers to a region of a DNA molecule that is desired to be amplified from a nucleic acid template. In an aspect, a target DNA sequence region of interest comprises one or more exon sequences. In an aspect, a target DNA sequence region of interest comprises one or more intron sequences. In an aspect, a target DNA sequence region of interest comprises one or more exon sequences and one or more intron sequences. In an aspect, a target DNA sequence region of interest comprises an intergenic region. [0141] In an aspect, a target DNA sequence region of interest consists of one or more exon sequences. In an aspect, a target DNA sequence region of interest consists of one or more intron sequences. In an aspect, a target DNA sequence region of interest consists of one or more exon sequences and one or more intron sequences. In an aspect, a target DNA sequence region of interest consists of an intergenic region.

[0142] In an aspect, a target DNA sequence region of interest comprises a cDNA molecule. In an aspect, a target DNA sequence region of interest comprises at least 20 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 50 nucleotides. In an aspect, a target

DNA sequence region of interest comprises at least 100 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 250 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 500 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 1000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 2000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 3000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 4000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 6000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 7000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 8000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 9000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 10,000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 11,000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 12,000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 13,000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 14,000 nucleotides. In an aspect, a target DNA sequence region of interest comprises at least 15,000 nucleotides.

[0143] In an aspect, a target DNA sequence region of interest comprises between 10 nucleotides and 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 20 nucleotides and 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 50 nucleotides and 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 100 nucleotides and 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 500 nucleotides and 5000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 10 nucleotides and 1000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 20 nucleotides and 1000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 50 nucleotides and 1000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 100 nucleotides and 1000 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 500 nucleotides and 1000 nucleotides.

In an aspect, a target DNA sequence region of interest comprises between 10 nucleotides and 500 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 20 nucleotides and 500 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 50 nucleotides and 500 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 100 nucleotides and 500 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 10 nucleotides and 100 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 20 nucleotides and 100 nucleotides. In an aspect, a target DNA sequence region of interest comprises between 50 nucleotides and 100 nucleotides.

[0144] In an aspect, a target DNA sequence comprising at least 1000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 2000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 3000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 4000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 5000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 6000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 7000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 8000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 9000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 10,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 11,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 12,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 13,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 14,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule. In an aspect, a target DNA sequence comprising at least 15,000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule.

[0145] In an aspect, tiling comprises the use of a plurality of OFPs and a plurality of IFPs. In an aspect, tiling comprises the use of a plurality of OFPs and a plurality of IFPs, where a gap is present between each OFP binding site and each IFP binding site.

[0146] In an aspect, a target DNA sequence region of interest is within a prokaryotic DNA molecule. In an aspect, a target DNA sequence region of interest is within a eukaryotic DNA molecule. In an aspect, a target DNA sequence region of interest is within a viral DNA molecule. In an aspect, a target DNA sequence region of interest is within a viroid DNA molecule.

[0147] In an aspect, a eukaryotic DNA molecule is selected from the group consisting of an animal DNA molecule, a plant DNA molecule, and a fungi DNA molecule. In an aspect, an animal DNA molecule is a human DNA molecule. In an aspect, a prokaryotic DNA molecule is selected from the group consisting of a bacteria DNA molecule and an archaea DNA molecule.

[0148] In an aspect, a virus DNA molecule is selected from the group consisting of an Adenoviridae DNA molecule, a Herpesviridae DNA molecule, a Poxviridae DNA molecule, a Papillomaviridae DNA molecule, a Parvoviridae DNA molecule, a Reoviridae DNA molecule, a Coronaviridae DNA molecule, a Picomaviridae DNA molecule, a Togaviridae DNA molecule, an Orthomyxoviridae DNA molecule, a Rhabdoviridae DNA molecule, a Retroviridae DNA molecule, a Hepadnaviridae DNA molecule, a Baculoviridae DNA molecule, a Geminiviridae DNA molecule, a Flaviviridae DNA molecule, a Filoviridae DNA molecule, a Paramyxoviridae DNA molecule, and a Pneumoviridae DNA molecule. In an aspect, a virus DNA molecule is selected from the group consisting of a human orthopnemovirus (HRSV)c DNA molecule, an influenza virus cDNA molecule, a human immunodeficiency virus (HIV) cDNA molecule, a hepatitis B virus (HBV) cDNA molecule, and a human papillomavirus (HPV) cDNA molecule.

[0149] In an aspect, a viroid DNA molecule is selected from the group consisting of a Pospiviroidae DNA molecule, and a Avsunviroidae DNA molecule.

[0150] As used herein, “Sample Barcode” or “SB” refers to unique sequences used to tag or track individual samples to allow for sample multiplexing and large numbers of libraries to be pooled and sequenced simultaneously during a single sequencing run. In an aspect, an LTA comprises an SB sequence. In an aspect, an SB is positioned in an LTA-top strand. In an aspect, an SB is positioned in an LTA-bottom strand. [0151] In an aspect, an LTA comprises a Unique Molecular Identifier sequence. As used herein, “Unique Molecular Identifier sequence” or “UMI” refers to molecular barcodes of short sequences which are used to uniquely tag each molecule in a sample library. Through UMI, each nucleic acid in a starting material is tagged with a unique molecular barcode. Sequencing with UMIs reduces the rate of false-positive variant calls and increase sensitivity of variant detection and quantification.

[0152] In an aspect, a UMI sequence comprises between 7 degenerate nucleotides and 30 degenerate nucleotides. In an aspect, a UMI sequence comprises between 5 degenerate nucleotides and 40 degenerate nucleotides. In an aspect, a UMI sequence comprises between 10 degenerate nucleotides and 20 degenerate nucleotides. In an aspect, a UMI sequence comprises at least 5 degenerate nucleotides. In an aspect, a UMI sequence comprises at least 7 degenerate nucleotides. In an aspect, a UMI sequence comprises at least 10 degenerate nucleotides. In an aspect, a UMI sequence comprises at least 15 degenerate nucleotides. In an aspect, a UMI sequence comprises fewer than 50 degenerate nucleotides. In an aspect, a UMI sequence comprises fewer than 40 degenerate nucleotides. In an aspect, a UMI sequence comprises fewer than 30 degenerate nucleotides. In an aspect, a UMI sequence comprises fewer than 20 degenerate nucleotides.

[0153] In an aspect, each degenerate nucleotide in a UMI sequence is selected from the group consisting of N, B, D, H, V, S, W, Y, R, M, and K.

[0154] In an aspect, a UMI sequence comprises between 7 degenerate nucleotides and 30 degenerate nucleotides, where each degenerate nucleotide is selected from the group consisting of N, B, D, H, V, S, W, Y, R, M, and K. In aspect, a UMI sequence comprises at least one degenerate nucleotide selected from the group consisting of R, Y, S, W, K, M, B, D, H, V, and N. In an aspect, a UMI sequence comprises a mixture of between 10 and 100 defined DNA sequences with a minimum pairwise Hamming distance of between 2 and 5. In an aspect, a UMI sequence comprises a mixture of between 10 and 1000 defined DNA sequences with a minimum pairwise Levenschtein distance of between 2 and 5.

[0155] In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 500 nM. In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, an IFP is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, an IFP is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, an IFP is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, an IFP is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, an IFP is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, an IFP is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, an IFP is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, an IFP is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, an IFP is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, an IFP is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, an IFP is present in a mixture at a concentration between 500 nM and 1000 nM.

[0156] In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 500 nM. In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, an OFP is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, an OFP is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, an OFP is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, an OFP is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, an OFP is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, an OFP is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, an OFP is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, an OFP is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, an OFP is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, an OFP is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, an OFP is present in a mixture at a concentration between 500 nM and 1000 nM.

[0157] In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 500 nM. In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, an MFP is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, an MFP is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, an MFP is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, an MFP is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, an MFP is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, an MFP is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, an MFP is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, an MFP is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, an MFP is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, an MFP is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, an MFP is present in a mixture at a concentration between 500 nM and 1000 nM.

[0158] In an aspect, a UP is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, a UP is present in a mixture at a concentration between 1 nM and 500 nM. In an aspect, a UP is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, a

UP is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, a UP is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, a UP is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, a UP is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, a UP is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, a UP is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, a UP is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, a UP is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, a UP is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, a UP is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, a UP is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, a UP is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, a UP is present in a mixture at a concentration between 500 nM and 1000 nM.

[0159] In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 500 nM.

In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, a blocker is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, a blocker is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, a blocker is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, a blocker is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, a blocker is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, a blocker is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, a blocker is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, a blocker is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, a blocker is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, a blocker is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, a blocker is present in a mixture at a concentration between 500 nM and 1000 nM.

[0160] In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 1000 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 500 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 250 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 100 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 50 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 1 nM and 10 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 50 nM and 1000 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 50 nM and 500 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 50 nM and 250 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 50 nM and 100 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 100 nM and 1000 nM. In an aspect, a wildtypespecific blocker is present in a mixture at a concentration between 100 nM and 500 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 100 nM and 250 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 250 nM and 1000 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 250 nM and 500 nM. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration between 500 nM and 1000 nM.

[0161] In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 5 times and 20 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 5 times and 15 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 5 times and 10 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 5 times and 7.5 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 7.5 times and 20 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 7.5 times and 15 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 7.5 times and 10 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 10 times and 20 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 10 times and 15 times higher than the concentration of an IFP in the mixture. In an aspect, a wildtype-specific blocker is present in a mixture at a concentration that is between 15 times and 20 times higher than the concentration of an IFP in the mixture.

[0162] In an aspect, this application provides a method for preparing a nucleic acid for sequencing, the method comprising: (i) ligating a Ligation Tail Adapter (LTA) molecule to a nucleic acid comprising a known target nucleotide sequence to produce a ligation product, where the LTA molecule comprises an LTA-top strand and an LTA-bottom strand, and where the LTA- top strand and the LTA-bottom strand which two strands form a Double-Stranded End (DSE) and a DNA Tail (DT) region to produce a ligation product; (ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a Splint, where the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region to produce an amplification product; and (iii) amplifying an amplification product of (ii) using a second target-specific primer that specifically anneals to the amplification product of (ii) and a Universal Primer (UP) comprising a sequence which can specifically anneal to the 5' end subsequence, or a portion thereof, of the Splint, where the second target-specific primer is nested relative to the first target-specific primer. In an aspect, the method further comprises mechanically shearing a nucleic acid molecule preparation to obtain a nucleic acid molecule prior to step (i). In an aspect, the method further comprises end-repairing a mechanically sheared nucleic acid molecule. In an aspect, the method further comprises phosphorylating a mechanically sheared nucleic acid molecule. In an aspect, the method further comprises subjecting an RNA molecule to a reverse transcriptase regimen to generate a DNA molecule (e.g, a cDNA molecule). As used here, a “reverse transcriptase regimen” refers to any protocol known in the art that uses a reverse transcriptase to generate a cDNA molecule from an RNA molecule. In an aspect, the method further comprises adenylating a nucleic acid molecule to produce a 3 '-adenosine overhang on the nucleic acid molecule, and where a DSE comprises a 3' thymine overhang prior to step (i).

[0163] In an aspect, ligating comprises performing an overhang ligating reaction. In an aspect, ligating comprises the use of a T4 DNA ligase enzyme. In an aspect, ligating comprises the use of a T3 DNA ligase enzyme. In an aspect, ligating comprises the use of a T7 DNA ligase enzyme. In an aspect, ligating comprises performing a TA ligation reaction.

[0164] As used herein, a “nucleic acid molecule preparation” refers to any substance or mixture that comprises a biological sample. As used herein, a “biological sample” refers to a material obtained or isolated from a fresh or preserved biological sample or synthetically created source that contains nucleic acids. Any nucleic acid molecule provided herein can be obtained from a biological sample. Biological samples, without being limiting, include at least one cell, fetal cell, cell culture, tissue specimen, blood, serum, plasma, saliva, urine, tear, vaginal secretion, sweat, lymph fluid, cerebrospinal fluid, mucosa secretion, peritoneal fluid, ascites fluid, fecal matter, body exudates, umbilical cord blood, chorionic villi, amniotic fluid, embryonic tissue, multicellular embryo, lysate, extract, solution, or reaction mixture suspected of containing nucleic acids. In an aspect, a biological sample is obtained from the environment (e.g., from soil, water, or air). In an aspect, a biological sample is obtained directly from an organism. In an aspect, a biological sample is obtained from a living organism. In an aspect, a biological sample is obtained from a deceased organism. In an aspect, a biological sample is obtained from a cell line. A biological sample can comprise DNA, RNA, or both. In an aspect, a biological sample is from a healthy organism. In an aspect, a biological sample is from a diseased organism. In an aspect, a biological sample is from a mutagenized sample. In an aspect, the nucleic acids obtained from a biological sample can be converted to cDNA.

[0165] In an aspect, a biological sample is a prokaryotic biological sample. In an aspect, a biological sample is a eukaryotic biological sample. In an aspect, a biological sample is an animal biological sample. In an aspect, a biological sample is a plant biological sample. In an aspect, a biological sample is a fungal biological sample. In an aspect, a biological sample is a protozoan biological sample. In an aspect, a biological sample is a mammalian biological sample. In an aspect, a biological sample is a primate biological sample. In an aspect, a biological sample is a human biological sample.

[0166] In an aspect, a second target-specific primer can specifically anneal to a portion of a known target nucleotide sequence within an amplification product. In an aspect, a known target nucleotide sequence comprises a sequence associated with a gene rearrangement.

[0167] In an aspect, this application provides a method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising: (a) ligating a target nucleic acid molecule comprising the known target nucleotide sequence with a universal Ligation Tail Adapter (LTA), where the universal LTA comprises a non-amplification strand and an amplification strand to produce a ligation product; (b) amplifying a portion of the target nucleic acid molecule and the amplification strand of the universal LTA with a Splint and a first targetspecific primer from the ligation product to produce a first amplicon; (c) amplifying a portion of the first amplicon with a Universal Primer (UP) and a second target-specific primer to produce a second amplicon; and (d) sequencing the second amplicon using a first sequencing primer and a second sequencing primer; where the universal LTA comprises a ligatable Double-Stranded End (DSE) and a DNA Tail (DT) region; where the non-amplification strand comprises a 5' duplex portion; where the amplification strand comprises an unpaired 5' portion, a 3' duplex portion, and a 3' thymine (T) overhang; where the duplex portion of the non-amplification strand and the duplex portion of the amplification strand are complementary and form the ligatable DSE comprising a 3' T overhang; where the duplex portion is of sufficient length to remain in duplex form at the ligation temperature; where the first target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid molecule; where the second target-specific primer comprises a 3' portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence within the first amplicon, and a 5' portion comprising a nucleic acid sequence that is identical to the second sequencing primer and the second target-specific primer is nested with respect to the first targetspecific primer.

[0168] In an aspect, this application provides a method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising: (i) ligating a universal Ligation Tail Adapter (LTA) to a nucleic acid molecule comprising the known target nucleotide sequence to produce a ligation product; (ii) amplifying the ligation product via polymerase chain reaction using a Splint that specifically anneals to the universal LTA, and a first target-specific primer that specifically anneals to the known target nucleotide sequence to produce a first amplification product; (iii) amplifying the first amplification product via polymerase chain reaction using a Splint-specific primer and a second target-specific primer, where the second target-specific primer is nested relative to the first target-specific primer to produce a second amplification product; and (iv) sequencing the second amplification product using a first sequencing primer and a second sequencing primer, where the first sequencing primer and the second sequencing primer are complementary to opposite strands of the second amplification product.

[0169] In an aspect, this application provides a method of determining if a subject in need of treatment for cancer will be responsive to a given treatment, the method comprising: detecting, in a tumor sample obtained from the subject, the presence of an oncogene rearrangement according to any method provided herein; where the subject is determined to be responsive to a treatment targeting the oncogene rearrangement product if the presence of the oncogene rearrangement is detected.

[0170] In an aspect, this disclosure provides a method of treating cancer, the method comprising: detecting, in a tumor sample obtained from a subject in need of treatment for cancer, the presence an oncogene rearrangement according to any method provided herein; and administering a cancer treatment which is effective against tumors comprising the oncogene rearrangement.

[0171] In an aspect, an oncogene rearrangement is an ALK oncogene rearrangement. In an aspect, an oncogene rearrangement is an ALK oncogene rearrangement, and where a subject will be responsive to a treatment selected from the group consisting of an ALK inhibitor; crizotinib (PF-02341066); AP26113; LDK378; 3-39; AF802; IPI-504; ASP3026; AP-26113; X-396; GSK- 1838705A; CH5424802; and NVP- TAE684.

[0172] In an aspect, an oncogene rearrangement is an ROS1 oncogene rearrangement. In an aspect, an oncogene rearrangement is an ROS1 oncogene rearrangement, and where a subject will be responsive to a treatment selected from the group consisting of an ALK inhibitor; crizotinib (PF-02341066); AP26113; LDK378; 3-39; AF802; IPI-504; ASP3026; AP-26113; X-396; GSK- 1838705A; CH5424802; and NVP- TAE684.

[0173] In an aspect, an oncogene rearrangement is an RET oncogene rearrangement. In an aspect, an oncogene rearrangement is an RET oncogene rearrangement, and where a subject will be responsive to a treatment selected from the group consisting of a RET inhibitor; DP -2490; DP- 3636; SU5416; BAY 43-9006; BAY 73-4506 (regorafenib); ZD6474; NVP-AST487; sorafenib; RPI-1; XL184; vandetanib; sunitinib; imatinib; pazopanib; axitinib; motesanib; gefitinib; and withaferin A.

[0174] In an aspect, a cancer is lung cancer. In an aspect, a cancer is selected from the group consisting of breast cancer, colorectal cancer, endometrial cancer, fallopian tube cancer, ovarian cancer, primary peritoneal cancer, gastric cancer, melanoma, pancreatic cancer, prostate cancer, sarcoma, carcinoma, leukemia, brain cancer, central nervous system cancer, adrenal cortex cancer, gallbladder cancer, urinary tract cancer, thyroid cancer, liver cancer, kidney cancer, eye cancer, and lymphoma.

[0175] US Patent Nos. 10,718,009 and 9,487,828 reported an anchored multiplex PCR assay attempting to amplify fusion DNA or RNA sequences by using two nested gene-specific primers on one end of the sequence. This approach is limited in that it proportionally enriches all areas in DNA sequences and cannot effectively detect DNA variants with a fraction lower than 1%. [0176] Here a technology is described to selectively enrich DNA variants on one side of the sequence near the variation region or site. We use a wildtype Blocker to suppress the amplification of DNA segments without variants, and preferably amplify DNA sequence variants. This method can be integrated with various downstream readout methods, including qPCR, Microarray, and sequencing. We primarily describe sequencing-related applications below. Next-generation sequencing (NGS) is a high-throughput approach that can massively sequence DNA in a short amount of time and will be the primary embodiment described below.

[0177] The LTA approach can be used for gene fusion detection. The coding sequence of a gene can be on either the (+) strand or the (-) strand of the human genome. Used herein, sequences are referenced based on the coding strand, with upstream defined as the sequence to the 5' end of the transcribed RNA sequence and downstream defined as the sequence to the 3' end of the transcribed RNA sequence.

[0178] Figure 1 shows a schematic illustration of an example of NGS library construction for targeted RNA and DNA sequencing using a ligation tail adapter (LTA). A DNA template molecule is first ligated to the LTA. The LTA comprises an LTA-top strand and an LTA-bottom strand. These two strands form a Double- Stranded End (DSE) for ligation with a nucleic acid template molecule, and a DNA Tail (DT) region. In some embodiments, a DT region comprises or is a single-stranded DNA, and in other embodiments a DT region comprises or is a double-stranded DNA. In some embodiments, an LTA comprises at least one single-stranded DT sequence in the LTA-top strand or the LTA-bottom strand, which cannot bind with each other. In some embodiments, a DT is a double-stranded DNA that comprises a UMI and/or an SB single-stranded DNA between the DSE and the DT region. In some embodiments, a DNA template contains genomic DNA. In some embodiments, a DNA template contains sheared genomic DNA. In some embodiments, a DNA template contains cDNA, which is produced from an RNA reverse transcription.

[0179] An LTA comprises DSE and DT regions (Figure 2). In some embodiments, a DSE has a length of 5 - 50 bp. In some embodiments, a DT comprises a double-stranded DNA. In some embodiments, a DT comprises a single-stranded DNA. In some embodiments, a DT comprises a double-stranded DNA with a top strand that has over 80%, 90%, and 100% percent identity to the reverse complement of the bottom strand. In some embodiments, a DT region comprises a singlestranded Unique Molecular Identifier (UMI) or a Sample Barcode (SB) between the DSE and the DT region. In some embodiments, the single-stranded DNA is in the LTA-top strand. In some embodiments, the single-stranded DNA is in the LTA-bottom strand. In some embodiments, the single-stranded DNA is in the LTA-top and the LTA-bottom strands. In some embodiments, the single-stranded DNA length ranges from 5 nt to 100 nt. In some embodiments, an LTA-top strand comprises a UMI or an SB between the DSE and a single-stranded DNA. In some embodiments, the UMI and the SB are in an LTA-bottom strand.

[0180] An outer PCR step occurs after a LTA is ligated to a DNA template, and comprises an OFP as a forward primer and a Splint as a reverse primer. In some embodiments, the number of PCR cycles ranges from 1 cycle to 40 cycles. In some embodiments, a Splint comprises the full- length reverse complement, or a part thereof, of the LTA-top strand and a unique 5' overhang that cannot bind to the LTA-top strand (Figure 4). In some embodiments, the length of the unique single-stranded end ranges from 1 nt to 100 nt. In some embodiments, a Splint binds to the DT region of a LTA-top strand from the second base of the DT 5' region to the 10 ^th nt of the 3' region. In some embodiments, a Splint binds to the DT starting from the first base of the 5' region of the DT. In some embodiments, a Splint binds to the LTA-top strand starting from the second base of the 5' region of the LTA-top strand to the second base of the 3' region of the DSE. In some embodiments, a Splint binds to a LTA-top strand starting from the first base of the 5' end of the LTA-top strand. In some embodiments, a Splint comprises a hairpin structure at the 5' end and a single-stranded DNA at the 3' end.

[0181] After an outer PCR step, the amplicon is purified or diluted and then used as a template for an inner PCR step. In some embodiments, an IFP binding site is immediately adjacent to an OFP binding site, which means the OFP and the IFP tile the same template without a gap or an overlap (Figure 5a). In some embodiments, an IFP binding site and an OFP binding site has a gap from 1-50 nt (Figure 5b). In some embodiments, an IFP binding site and an OFP binding site (e.g., the IFP 5' end and the OFP 3' end) has an overlap from l-40nt (Figure 5c). A UP sequence serves as a reverse primer that contains the same region of a Splint (Figure 6). In some embodiments, a UP primer comprises a reverse complement of part of the outer PCR amplicon positive strand and a unique 5' overhang that cannot bind to the outer PCR amplicon (UP-1). In some embodiments, a UP is the same as the 5' overhang of the Splint (UP -2). In some embodiments, a UP comprises a reverse complement of a DT in the LTA-top strand and a 5' overhang region of a Splint (UP-3). In some embodiments, the number of the inner PCR cycles are between 1 cycle to 40 cycles. After the inner PCR, the amplicon is defined as the Nested PCR Amplicon.

[0182] In some embodiments, index primers can be added to both sides of the Nested PCR Amplicon for paired end sequencing in an NGS platform (Figure 7). An adapter PCR comprises an inner AD-FP as a forward primer and a UP as a reverse primer. An inner AD-FP comprises adapter sequences in the 5' end and an IFP sequence in the 3' end. In some embodiments, an index PCR comprises a sequencing index 1 as a forward primer and a sequencing index 2 as a reverse primer. In some embodiments, the sequencing index 1 is an Illumina P5 index primer, and the sequencing index 2 is an illumine P7 index primer. In some embodiments, the sequencing index only contains one side for single-end sequencing. In some embodiments, the sequencing index can be a nanopore primer for nanopore sequencing. In some embodiments, the sequencing index can be an adapter for Ion Torrent sequencing.

[0183] In some embodiments, an UMI or SB addition is prepared following the protocol in Figure 1 if the UMI or SB is in the LTA-top strand. In some embodiments, a UMI or SB in the LTA-bottom strand and will be added follow the workflow shown in Figure 14. In some embodiments, after ligation, a UMI-containing forward primer is added in a PCR reaction for 1 cycle of PCR to add the UMI. A library can then be prepared following the outer PCR, the inner PCR and the index PCR.

[0184] In some embodiments, the inner PCR can be combined with a Blocker Displacement Amplification (BDA) (Figure 13) and the overall workflow was named as Adjacent Blocker Displacement Amplification (ABDA). BDA was previously described in US20170067090 and WO2019164885. The ligation and outer PCR steps are followed by an adjacent PCR step. The BDA step comprises an IFP, a Blocker, and a UP primer as the reverse primer.

[0185] An IFP and a Blocker are designed to have a certain degree of sequence overlap with several nucleotides at the 3' end. A 3' region of each IFP sequence is identical in sequence to a 5' region of the corresponding Blocker. In some embodiments, this region has a length between 5 nt and 10 nt. In other embodiments, this region has a length between 3 nt and 25 nt. The binding of the IFP or the Blocker to the template will be mutually exclusive: with high probability (without being bound by any theory), a three-stranded molecule comprising the template, the IFP, and the Blocker colocalized via DNA hybridization interactions. The three-stranded molecule will rapidly dissociate, releasing a single-stranded forward primer or single-stranded Blocker into the solution. In some embodiments, the number of overlapping nucleotides of overlaps between the forward primer and the Blocker is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, the standard free energy of binding of the overlapping nucleotides to bind to their reverse complementary sequences is -4 kcal/mol at approximately 50 °C, approximately 55 °C, approximately 60 °C, approximately 65 °C, or approximately 70 °C in a buffer suitable for PCR.

[0186] In some embodiments, the binding of a Blocker to its reverse complementary sequence has a computed melting temperature of approximately 55 °C, approximately 60 °C, approximately 65 °C, approximately 70 °C, approximately 75 °C, or approximately 80 °C in a buffer suitable for PCR, at Blocker concentrations of between 100 nM and 5 pM. In some embodiments, the binding of a Blocker to its reverse complementary sequence has a computed standard free energy of binding (AG°) of approximately -14 kcal/mol at approximately 50 °C, approximately 55 °C, approximately 60 °C, approximately 65 °C, or approximately 70 °C in a buffer suitable for PCR.

[0187] In some embodiments, the standard free energy of a Blocker binding to its reverse complement (AG°B) is stronger than the standard free energy of a forward primer to bind to its reverse complement (AG°fp) by between -1 kcal/mol and -4 kcal/mol at approximately 50 °C, approximately 55 °C, approximately 60 °C, approximately 65 °C, or approximately 70 °C in abuffer suitable for PCR. In some embodiments, a Blocker comprises a sequence at or near the 3' end that does not hybridize to the template and prevents DNA polymerase extension. In some embodiments, a Blocker comprised a chemical modification at or near the 3' end that prevented DNA polymerase extension. In some embodiments, the Blocker comprises a chemical modification at or near the 3' end that prevents 3' ->5' exonuclease activity by error-correct DNA polymerases. In some embodiments, the chemical modification comprises inverted DNA nucleotides. In some embodiments, the chemical modification comprises 3-carbon spacers.

[0188] After the BDA step, two cycles of adapter PCR are used for adding the adapter of the IFP. Following the index PCR step, the index sequences for the Illumina NGS sequencing was added.

[0189] In an aspect, for any primer or Splint molecules, a template binding sequence comprises a length of between 5 nucleotides and 100 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 75 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 50 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 40 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 30 nucleotides. In an aspect, a template binding sequence comprises a length of between 6 nucleotides and 20 nucleotides.

[0190] In an aspect, for any primer or Splint molecules, a template binding sequence comprises a length of at least 5 nucleotides. In an aspect, a template binding sequence comprises a length of at least 6 nucleotides. In an aspect, a template binding sequence comprises a length of at least 10 nucleotides. In an aspect, a template binding sequence comprises a length of at least 15 nucleotides. In an aspect, a template binding sequence comprises a length of at least 20 nucleotides. In an aspect, a template binding sequence comprises a length of at least 25 nucleotides. In an aspect, a template binding sequence comprises a length of at least 30 nucleotides. In an aspect, a template binding sequence comprises a length of at least 40 nucleotides. In an aspect, a template binding sequence comprises a length of at least 50 nucleotides. In an aspect, a template binding sequence comprises a length of at least 75 nucleotides. [0191] In an aspect, this disclosure provides a template nucleic acid molecule. As used herein, a “template nucleic acid molecule” or a “template nucleic acid” refers to a nucleic acid molecule that comprises a sequence that is desired to be detected and/or amplified using PCR-based techniques in conjunction with at least one Occlusion Primer and/or at least one Occlusion Probe. In an aspect, a system comprises at least one template nucleic acid molecule. Any nucleic acid molecule that is desired to be detected or amplified can serve as a suitable template nucleic acid molecule. Numerous potential template nucleic acid molecules can be found in publicly available databases such as GenBank®. See Nucleic Acids Research, 41:D36-42 (2013).

[0192] In an aspect, a template nucleic acid molecule is a DNA molecule. In an aspect, a template nucleic acid molecule is an RNA molecule. In an aspect, a template nucleic acid molecule is a genomic DNA molecule. In an aspect, a template nucleic acid molecule is an organellar DNA molecule. In an aspect, an organellar DNA molecule is selected from the group consisting of a mitochondrial DNA molecule and a plastid DNA molecule. In an aspect, a template nucleic acid molecule is a complementary DNA (cDNA) molecule.

[0193] In an aspect, a template nucleic acid molecule is a eukaryotic nucleic acid molecule. In an aspect, a eukaryotic nucleic acid molecule is selected from the group consisting of an animal nucleic acid molecule, a plant nucleic acid molecule, and a fungi nucleic acid molecule. In an aspect, an animal nucleic acid molecule is a human nucleic acid molecule. In an aspect, a template nucleic acid molecule is a prokaryotic nucleic acid molecule. In an aspect, a prokaryotic nucleic acid molecule is selected from the group consisting of a bacteria nucleic acid molecule and an archaea nucleic acid molecule.

[0194] In an aspect, a template nucleic acid molecule is a virus nucleic acid molecule. In an aspect, a virus nucleic acid molecule is selected from the group consisting of an Adenoviridae nucleic acid molecule, a Herpesviridae nucleic acid molecule, a Poxviridae nucleic acid molecule, a Papillomaviridae nucleic acid molecule, a Parvoviridae nucleic acid molecule, a Reoviridae nucleic acid molecule, a Coronaviridae nucleic acid molecule, a Picomaviridae nucleic acid molecule, a Togaviridae nucleic acid molecule, an Orthomyxoviridae nucleic acid molecule, a Rhabdoviridae nucleic acid molecule, a Retroviridae nucleic acid molecule, a Hepadnaviridae nucleic acid molecule, a Baculoviridae nucleic acid molecule, a Geminiviridae nucleic acid molecule, a Flaviviridae nucleic acid molecule, a Filoviridae nucleic acid molecule, a Paramyxoviridae nucleic acid molecule, and a Pneumoviridae nucleic acid molecule. In an aspect, a virus nucleic acid molecule is selected from the group consisting of a human orthopnemovirus (HRSV) nucleic acid molecule, an influenza virus nucleic acid molecule, a human immunodeficiency virus (HIV) nucleic acid molecule, a hepatitis B virus (HBV) nucleic acid molecule, and a human papillomavirus (HPV) nucleic acid molecule.

[0195] In an aspect, a template nucleic acid molecule is a viroid nucleic acid molecule. In an aspect, a viroid nucleic acid molecule is selected from the group consisting of a Pospiviroidae nucleic acid molecule, and a Avsunviroidae nucleic acid molecule.

[0196] The following non-limiting embodiments are specifically envisioned:

1. A method of preparing a nucleic acid template into a DNA library for sequencing, the method comprising the steps of:

(a) Mixing a nucleic acid template, a Ligation Tail Adapter (LTA) molecule, a DNA ligase, and reagents for DNA ligase activity to form a first mixture; wherein the LTA molecule comprises an LTA-top strand and an LTA-bottom strand which two strands form a Double-Stranded End (DSE) and a DNA Tail (DT) region;

(b) Subjecting the first mixture to a suitable temperature to allow for DNA ligation to form a ligation product mixture;

(c) Introducing to the ligation product mixture from step (b) a target-specific Outer Forward Primer (OFP) and a Splint, a DNA polymerase, and reagents for DNA polymerase activity to form a second mixture; wherein the OFP comprises a nucleic acid sequence that can specifically anneal to a portion of a target DNA sequence region of interest, wherein the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region or portion thereof;

(d) Subjecting the second mixture to a suitable temperature to allow for DNA polymerase extension to form a DNA polymerase extension product mixture;

(e) Introducing to the DNA polymerase extension product mixture from step (d) a targetspecific Inner Forward Primer (IFP), a Universal Primer (UP), a thermostable DNA polymerase, and reagents for DNA polymerase activity to form a third mixture, wherein the IFP comprises a nucleic acid sequence that can specifically anneal to a portion of the target DNA sequence region of interest; and wherein the UP comprises a sequence which can specifically anneal to the 5' end subsequence, or portion thereof, of the Splint; and

(I) Subjecting the third mixture to temperature cycles to allow for polymerase chain reaction (PCR) amplification.

2. The method of embodiment 1, wherein the UP contains no sequence that is complementary to any 19 nucleotide or longer continuous subsequence of the LTA.

3. The method of embodiment 1, wherein the IFP is nested with respect to the OFP. 4. The method of embodiment 1, wherein the IFP and the OFP have a partially or completely overlapping binding site on the target DNA sequence region of interest.

5. The method of embodiment 1, wherein the 3' end subsequence of the Splint can specifically anneal to the DT region or portion thereof

6. The method of embodiment 1, wherein the LTA comprises a Sample Barcode (SB) sequence.

7. The method of any one of embodiments 1 - 6, wherein the SB is in the LTA-top strand.

8. The method of any one of embodiments 1 - 6, wherein the SB is in the LTA-bottom strand.

9. The method of any one of embodiments 1 - 8, wherein the LTA comprises a Unique Molecular Identifier (UMI) sequence.

10. The method of embodiment 9, wherein the UMI is in the LTA-top strand.

11. The method of embodiment 9, wherein the UMI is in the LTA-bottom strand.

12. The method of embodiment 9, wherein the UMI and SB are in the same LTA strand.

13. The method of embodiment 9, wherein the UMI sequence is from a mixture of 10 to 1000 defined DNA sequences with a minimum pairwise Hamming distance of 2, 3, 4, or 5.

14. The method of embodiment 9, wherein the UMI sequence is from a mixture of 10 to 1000 defined DNA sequences with a minimum pairwise Levenschtein distance of 2, 3, 4, or 5.

15. The method of embodiment 9, wherein the UMI sequence comprises at least one degenerate nucleotide selected from the group consisting of N, B, D, H, V, S, W, Y, R, M, and K..

16. The method of any one of embodiments 1 - 15, wherein the DNA polymerase used in step (b) is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase, phi29 DNA polymerase, KI enow fragment, Bst DNA polymerase, T4 DNA polymerase, Vent® DNA polymerase, LongAmp® Taq DNA polymerase, and OneTaq® DNA polymerase.

17. The method of any one of embodiments 1 - 16, wherein the DNA polymerase used in step (c) is selected from the group consisting of Taq DNA polymerase, Phusion® DNA polymerase, Q5® DNA polymerase, and KAPA High Fidelity DNA polymerase.

18. The method of any one of embodiments 1 - 17, wherein the LTA-top strand comprises a phosphorylated nucleotide at its 5' end, and the LTA-bottom strand comprises a thymine at its 3' end.

19. The method of any one of embodiments 1 - 18, wherein second nucleotide position from the 3' end of the LTA-bottom strand comprises a phosphorothioate bond modification.

20. The method of any one of embodiments 1 - 19, wherein the forward strand of the DSE begins from the 5' end of the LTA top strand, and the reverse strand of the DSE begins from the second nucleotide position from the 3' end of the LTA-bottom strand. 21. The method of any one of embodiments 1 - 20, wherein the DSE is a double-stranded DNA comprising a length of between 5 nucleotides and 90 nucleotides.

22. The method any one of embodiments 1 - 21, wherein the percent identity between the LTA- top strand and the reverse complementary of the LTA-bottom strand in the DSE region is at least 95%.

23. The method of any one of embodiments 1 - 22, wherein the DT region comprises a singlestranded DNA or a double-stranded DNA.

24. The method of any one of embodiments 1 - 22, wherein the LTA-top strand comprises a singlestranded DT region that does not bind to the LTA-bottom strand, and the LTA-bottom strand 5' region binds to the LTA-top strand to form a DSE.

25. The method of any one of embodiments 1 - 22, wherein the LTA-bottom strand comprises a single-stranded DT region that does not bind to the LTA-top strand, and the LTA-top strand 3' region binds to the LTA-bottom strand without any overhang bases to form a DSE.

26. The method of any one of embodiments 1 - 22, wherein both the LTA-top strand 3' region and the LTA-bottom strand 5' region comprise a single-stranded DT region that does not bind with each other; and wherein the LTA-top strand 5' region and the LTA-bottom strand 3' region binds with each other to form a DSE.

27. The method of any one of embodiments 1 - 22, wherein the LTA comprises, in the LTA-top or LTA-bottom strand, a single-stranded UMI and/or SB between the DSE and the DT region.

28. The method of any one of embodiments 1 - 22, wherein the DT region comprises a doublestranded sequence comprising between 1 and 30 nucleotide mismatches.

29. The method of any one of embodiments 1 - 28, wherein the nucleic acid template is a doublestranded DNA.

30. The method of any one of embodiments 1 - 29, wherein the nucleic acid template is a biological DNA derived from a sample of cells from biofluids such as blood, urine, saliva, cerebrospinal fluid, interstitial fluid, and synovial fluid, or from a tissue such as a biopsy tissue or a surgically resected tissue.

31. The method of any one of embodiments 1 - 29, wherein the nucleic acid template is a cDNA molecule generated through the reverse transcription of an RNA sample.

32. The method of embodiment 31 , wherein the RNA sample is a biological RNA sample derived from a human, an animal, a plant, or an environmental specimen.

33. The method of any one of embodiments 1 - 29, wherein the nucleic acid template is an amplicon DNA molecule generated through a DNA polymerase. 34. The method of any one of embodiments 1 - 33, wherein the nucleic acid template is from a physically, chemically, or enzymatically treated product of a biological DNA or RNA sample.

35. The method of any one of embodiments 1 - 33, wherein the nucleic acid template is from a product of a fragmentation process.

36. The method of embodiment 35, wherein the fragmentation process comprises ultrasonication or enzymatic fragmentation.

37. The method of any one of embodiments 1 - 36, wherein the nucleic acid template undergoes an end-repair process before Step (a).

38. The method of embodiment 37, the end-repair process is performed using a T4 DNA ligase enzyme.

39. The method of any one of embodiments 1 - 38, wherein the nucleic acid template is ligated using a blunt TA ligase enzyme.

40. The method of any one of embodiments 1 - 39, wherein the ligation product is purified after Step (a).

41. The method of embodiment 40, wherein the purification is selected from the group consisting of column purification, beads purification, diluting the ligation product between 10-fold and 10000-fold, and any combination thereof.

42. The method of embodiment 41, wherein the ligation product is diluted at least 20-fold.

43. The method of embodiment 41, wherein the ligation product is diluted equal to or less than 9000-fold.

44. The method of any one of embodiments 1 - 41, wherein the OFP comprises between 10 nucleotides and 100 nucleotides.

45. The method of any one of embodiments 1 - 44, wherein the OFP comprises a 5' overhang which does not bind to the reverse strand of the template sequence.

46. The method of embodiment 45, the 5' overhang in the OFP comprises between 1 nucleotide and 100 nucleotides.

47. The method of any one of embodiments 1 - 46, the OFP anneals to the template at a temperature between 55°C and 72°C.

48. The method of any one of embodiments 1 - 47, wherein the Splint comprises a 5' sequence that does not bind with the LTA, and wherein the 5' sequence comprises a length between 1 nucleotide and 100 nucleotides.

49. The method of embodiment 48, wherein the Splint 5' sequence comprises at least 3nucleotides.

50. The method of embodiment 48, wherein the Splint 5' sequence comprises less than or equal to 90 nucleotides. 51. The method of embodiment 48, wherein the Splint 5' sequence comprises between 3 nucleotides and 5 nucleotides.

52. The method of embodiment 48, wherein the Splint 5' sequence comprises between 15 nucleotides and 50 nucleotides.

53. The method of embodiment 48, wherein the Splint comprises a 3' sequence capable of binding to a single-stranded 3' overhang of the LTA-top strand from the second nucleotide position following the DSE to at least the 10 ^th nucleotide position on the 3' end of the DT region.

54. The method of embodiment 48, wherein the Splint comprises a 3' sequence capable of binding to a 3' overhang of the LTA-top strand from the first nucleotide position following the DSE.

55. The method of embodiment 48, wherein the Splint comprises a 3' sequence capable of binding to the DSE region starting from the second nucleotide position on the 5' end of the LTA-top strand to the second nucleotide position of the 3' end of the DSE.

56. The method of embodiment 48, wherein the Splint comprises a 3' sequence capable of binding to the DSE region starting from the first nucleotide position of the 5' sequence of the LTA-top strand.

57. The method of embodiment 48, wherein the Splint binds to the LTA-bottom strand instead of the LTA-top strand.

58. The method of embodiment 48, wherein the Splint comprises, in order from 5' to 3' end, a first sequence, a second sequence, a third sequence, and a fourth sequence, wherein the third sequence is complementary to the first sequence.

59. The method of any one of embodiments 1 - 58, wherein the IFP comprises between 10 nucleotides and 70 nucleotides.

60. The method of embodiment 59, wherein the IFP comprises at least 12nucleotides.

61. The method of embodiment 59, wherein the IFP comprises less than or equal to 60 nucleotides.

62. The method of embodiment 59, wherein the IFP comprises between 20 nucleotides and 25 nucleotides.

63. The method of embodiment 59, wherein the IFP comprises between 20 nucleotides and 30 nucleotides.

64. The method of any one of embodiments 1 - 63, wherein the IFP anneals to the template at a temperature from 55°C to 72°C.

65. The method of any one of embodiments 1 - 64, wherein the IFP comprises a 5'-end singlestranded nucleic acid sequence which does not bind to the nucleic acid template.

66. The method of embodiment 65, wherein the 5' end single-stranded nucleic acid sequence comprises a sequencing adapter. 67. The method of embodiment 66, wherein the sequencing adapter is or is a part of an Illumina sequencing adapter, a nanopore sequencing adapter, or an Ion Torrent adapter.

68. The method of any one of embodiments 1-67, wherein the IFP binds a template nucleic acid at an IFP binding site that is 5' relative to an OFP binding site, and wherein the IFP and the OFP do not overlap when bound to the template nucleic acid.

69. The method of embodiment 68, wherein the IFP binding site overlaps with the OFP binding site to form an overlapping region, and wherein the overlapping region comprises between 1 nucleotide and 40 nucleotides.

70. The method of embodiment 69, wherein the overlapping region comprises at least 3 nucleotides.

71. The method of embodiment 69, wherein the overlapping region comprises less than or equal to 35 nucleotides.

72. The method of embodiment 69, wherein the overlapping region comprises between 3 nucleotides and 5 nucleotides.

73. The method of embodiment 69, wherein the overlapping region comprises between 5 nucleotides and 15 nucleotides.

74. The method of embodiment 68, wherein the OFP binding site and the IFP binding site are separated by a gap, and wherein gap comprises between 1 nucleotide and 50 nucleotide.

75. The method of embodiment 74, wherein the gap comprises least 3 nucleotides.

76. The method of embodiment 74, wherein the gap comprises less than or equal to 45 nucleotides.

77. The method of embodiment 74, wherein the gap comprises between 3 nucleotides and 5 nucleotides.

78. The method of embodiment 74, wherein the gap comprises between 3 nucleotides and 10 nucleotides.

79. The method of embodiment 68, wherein the OFP binding site and the IFP binding site are adjacent.

80. The method of any one of embodiments 1 - 79, wherein the OFP and the IFP provide a nested polymerase chain reaction (PCR) that further comprises a middle PCR to improve the specificity and on-target rate.

81. The method of embodiment 80, wherein the middle PCR comprises using an MFP that binds to an MFP binding site on at least one template nucleic acid molecule, wherein the MFP binding site partially overlaps with the OFP binding site. 82. The method of any one of embodiments 80 - 81, wherein the middle PCR comprises using an MFP that binds to an MFP binding site on at least one template nucleic acid molecule, wherein the MFP binding site partially overlaps with the IFP binding site.

83. The method of embodiment 80, wherein the middle PCR comprises using an MFP that comprises a 5' region starting from the second nucleotide of the OFP 5' region to the second nucleotide of the OFP 3' region, and the MFP comprises a 3' region starting from the second nucleotide of the IFP 5' region to the second nucleotide of the IFP 3' region.

84. The method of any one of embodiments 80 - 83, wherein the MFP comprises between 10 nucleotides and 70 nucleotides.

85. The method of embodiment 84, wherein the MFP comprises at least 12 nucleotides.

86. The method of embodiment 84, wherein the MFP comprises less than or equal to 60 nucleotides.

87. The method of embodiment 84, wherein the MFP comprises between 10 nucleotides and 15 nucleotides.

88. The method of embodiment 84, wherein the MFP comprises between 15 nucleotides and 25 nucleotides.

89. The method of any one of embodiments 1 - 88, wherein the UP comprises betweenlO nucleotides and 70 nucleotides.

90. The method of embodiment 89, wherein the UP comprises at least 12 nucleotides.

91. The method of embodiment 89, wherein the UP comprises less than or equal to 60 nucleotides.

92. The method of embodiment 89, wherein the UP comprises between 10 nucleotides and 15 nucleotides.

93. The method of embodiment 89, wherein the UP comprises between 15 nucleotides and 25 nucleotides.

94. The method of any one of embodiments 1 - 93, wherein the UP comprises a sequencing adapter.

95. The method of embodiment 94, wherein the sequencing adapter comprises a sequencing adapter selected from the group consisting of an Illumina sequencing adapter, a nanopore sequencing adapter, and an Ion Torrent sequencing adapter.

96. The method of any one of embodiments 1 - 89, wherein the UP comprises a partial sequencing adapter which is amplified with a sequencing index primer.

97. The method of any one of embodiments 1 - 96, wherein the DNA polymerase extension of step (d), the PCR amplification of step (f), or both, are multiplexed.

98. The method of any one of embodiments 1- 96, wherein a plurality of OFPs or a plurality of IFPs are used together. 99. The method of any one of embodiments 1 - 98, wherein the target DNA sequence region of interest comprises one or more exon sequences.

100. The method of any one of embodiments 1 - 98, wherein the target DNA sequence region of interest comprises one or more intron sequences.

101. The method of any one of embodiments 1 - 98, wherein the target DNA sequence region of interest comprises an intergenic region.

102. The method of any one of embodiments 1 - 98, wherein an amplicon amplified from the target DNA sequence region comprises at least 25 nucleotides.

103. The method of any one of embodiments 1 -102, wherein a target DNA sequence region comprising at least 1000 nucleotides is tiled from a first orientation based on the positive strand of the template nucleic acid molecule and a second orientation based on the negative strand of the templated nucleic acid molecule.

104. The method of embodiment 103, wherein the tiling comprises the use of a plurality of OFPs and a plurality of IFPs, wherein where a gap is present between each OFP binding site and each IFP binding site.

105. The method of embodiment 104, wherein the gap comprises at least 12 nucleotides.

106. The method of embodiment 104, wherein the gap comprises less than or equal to 60 nucleotides.

107. The method of embodiment 104, wherein the gap comprises between 10 nucleotides and 15 nucleotides.

108. The method of embodiment 104, wherein the gap comprises between 15 nucleotides and 50 nucleotides.

109. The method of any one of embodiments 1 - 108, wherein a binding site of the IFP is separated from a breakpoint of a known template region by a gap distance of between 5 nucleotides and 30 nucleotides.

110. The method of embodiment 109, wherein the gap distance comprises at least 7 nucleotides.

111. The method of embodiment 109, wherein the gap distance comprises less than or equal to 25 nucleotides.

112. The method of embodiment 109, wherein the gap distance comprises between 5 nucleotides and 7 nucleotides.

113. The method of embodiment 109, wherein the gap distance comprises between 15 nucleotides and 25 nucleotides. . The method of any one of embodiments 1 - 113, wherein the PCR amplification comprises a wildtype-specific Blocker. . The method of embodiment 114, wherein the wildtype-specific blocker binds to the target DNA sequence region of interest at a wildtype-specific binding site, wherein the IFP binds to the target DNA sequence region of interest at an IFP binding site, and wherein the wildtype-specific binding site and the IFP binding site overlap by at least 3 nucleotides.. The method of embodiment 114, wherein the wildtype-specific blocker binds to the target DNA sequence region of interest at a wildtype-specific binding site, wherein the IFP binds to the target DNA sequence region of interest at an IFP binding site, and wherein the wildtype-specific binding site and the IFP binding site overlap by less than or equal to 25 nucleotides. . The method of embodiment 114, wherein the wildtype-specific blocker binds to the target DNA sequence region of interest at a wildtype-specific binding site, wherein the IFP binds to the target DNA sequence region of interest at an IFP binding site, and wherein the wildtype-specific binding site and the IFP binding site overlap by between 3 nucleotides and 5 nucleotides. . The method of embodiment 114, wherein the wildtype-specific blocker binds to the target DNA sequence region of interest at a wildtype-specific binding site, wherein the IFP binds to the target DNA sequence region of interest at an IFP binding site, and wherein the wildtype-specific binding site and the IFP binding site overlap by between 5 nucleotides and 25 nucleotides. . The method of embodiment 114, wherein the IFP comprises a target-specific portion that does not overlap in sequence with the wildtype-specific blocker, wherein the wildtypespecific blocker comprises a blocker-unique sequence that does not overlap with the IFP, and where the IFP comprises an overlapping region that overlaps in sequence with the wildtype- specific blocker, and wherein:

(a) the overlapping region comprises a standard free energy of binding between -2 kcal/mol and -4 kcal/mol;

(b) the target-specific portion comprises a standard free energy of binding between -5 kcal/mol and -9 kcal/mol; and

(c) the blocker-unique sequence has a standard free energy of binding between -7 kcal/mol and -12 kcal/mol. . The method of embodiment 114, wherein the IFP comprises a target-specific portion that does not overlap in sequence with the wildtype-specific blocker, wherein the wildtype- specific blocker comprises a blocker-unique sequence that does not overlap with the IFP, and where the IFP comprises an overlapping region that overlaps in sequence with the wildtypespecific blocker, and wherein the overlapping region comprises standard free energy of binding that ranges between -2 kcal/mol and -4 kcal/mol.

121. The method of embodiment 120, wherein the target-specific portion comprises a standard free energy of binding between -5 kcal/mol and -9 kcal/mol.

122. The method of embodiment 120 or 121, wherein the blocker-unique sequence comprises a standard free energy of binding between -7 kcal/mol and -12 kcal/mol.

123. The method of any one of embodiments 114 - 122, wherein the wildtype-specific blocker comprises a terminator to prevent 3' to 5' DNA polymerase exonuclease activity, wherein the terminator is selected from the group consisting of a three-carbon (C3) spacer and DXXDM, wherein D is a match between the wildtype-specific blocker sequence and the target DNA region, wherein M is a C3 spacer, and wherein X is a mismatch between the wildtypespecific blocker sequence and the target DNA region.

124. The method of any one of embodiments 114 - 122, wherein the wildtype-specific blocker comprises a terminator comprising a DNA overhang comprising four nucleotides.

125. The method of any one of embodiments 114 - 122, wherein:

(a) the IFP is present at a concentration between 1 nM and 1000 nM; or

(b) the UP primer is present at a concentration between 1 nM and 1000 nM; or,

(c) the wildtype-specific blocker is present at a total concentration that is between 5 times and 20 times higher than the concentration of the IFP; or

(d) any combination of (a), (b), and (c).

126. A method for preparing a nucleic acid for sequencing, the method comprising:

(i) ligating a Ligation Tail Adapter (LTA) molecule to a nucleic acid molecule comprising a known target nucleotide sequence to produce a ligation product, wherein the LTA molecule comprises an LTA-top strand and an LTA-bottom strand, and wherein the LTA-top strand and the LTA-bottom strand form a Double-Stranded End (DSE) and a DNA Tail (DT) region to produce a ligation product;

(ii) amplifying the ligation product using a first target-specific primer that specifically anneals to the known target nucleotide sequence and a Splint, wherein the Splint comprises a 5' end subsequence that is not complementary to a subsequence of the LTA, and a 3' end subsequence that is complementary to the DT region to produce an amplification product; and

(iii) amplifying the amplification product using a second target-specific primer that specifically anneals to the amplification product and a Universal Primer (UP) comprising a sequence which can specifically anneal to the 5' end subsequence, or a portion thereof, of the Splint, wherein the second target-specific primer is nested relative to the first target-specific primer.

127. The method of embodiment 126, wherein the second target-specific primer can specifically anneal to a portion of the known target nucleotide sequence within the amplification product.

128. The method of embodiment 126 or 127, wherein the nucleic acid molecule is a deoxyribonucleic acid (DNA) molecule.

129. The method of embodiment 128, wherein the method further comprises subjecting a ribonucleic acid molecule to a reverse transcriptase regimen to generate the DNA molecule.

130. The method of any one of embodiments 126-128, wherein the method further comprises mechanically shearing a nucleic acid molecule preparation to obtain the nucleic acid molecule prior to step (i).

131. The method of embodiment 130, wherein the method further comprises end-repairing the nucleic acid molecule.

132. The method of embodiment 130, wherein the method further comprises phosphorylating the nucleic acid molecule.

133. The method of any one of embodiments 126-132, wherein the method further comprises adenylating the nucleic acid molecule to produce a 3 '-adenosine overhang on the nucleic acid molecule, and wherein the DSE comprises a 3' thymine overhang, prior to step (i).

134. The method of any one of embodiments 126-133, wherein the ligating comprises performing an overhang ligation reaction.

135. The method of any one of embodiments 126-134, wherein the ligating comprises performing a TA ligation reaction.

136. The method of any one of embodiments 126-135, wherein the first target-specific primer anneals to the known target nucleotide sequence at an annealing temperature between 61°C and 72°C.

137. The method of any one of embodiments 126-136, wherein the known target nucleotide sequence comprises a sequence associated with a gene rearrangement.

138. The method of any one of embodiments 126-138, wherein the LTA further comprises a sample barcode.

139. A method of determining the nucleotide sequence contiguous to a known target nucleotide sequence, the method comprising: (a) ligating a target nucleic acid molecule comprising the known target nucleotide sequence with a universal Ligation Tail Adapter (LTA), wherein the universal LTA comprises a nonamplification strand and an amplification strand to produce a ligation product;

(b) amplifying a portion of the target nucleic acid molecule and the amplification strand of the universal LTA with a Splint and a first target-specific primer from the ligation product to produce a first amplicon;

(c) amplifying a portion of the first amplicon with a Universal Primer (UP) and a second targetspecific primer to produce a second amplicon; and

(d) sequencing the second amplicon using a first sequencing primer and a second sequencing primer; wherein the universal LTA comprises a ligatable Double-Stranded End (DSE) and a DNA Tail (DT) region; wherein the non-amplification strand comprises a 5' duplex portion; wherein the amplification strand comprises an unpaired 5' portion, a 3' duplex portion, and a 3' thymine (T) overhang; wherein the duplex portion of the non-amplification strand and the duplex portion of the amplification strand are complementary and form the ligatable DSE comprising a 3' T overhang; wherein the duplex portion is of sufficient length to remain in duplex form at the ligation temperature; wherein the first target-specific primer comprises a nucleic acid sequence that can specifically anneal to the known target nucleotide sequence of the target nucleic acid molecule; wherein the second target-specific primer comprises a 3' portion comprising a nucleic acid sequence that can specifically anneal to a portion of the known target nucleotide sequence within the first amplicon, and a 5' portion comprising a nucleic acid sequence that is identical to the second sequencing primer and the second target-specific primer is nested with respect to the first target-specific primer. . A method for determining the nucleotide sequence contiguous to a known target nucleotide sequence of 10 or more nucleotides, the method comprising:

(i) ligating a universal Ligation Tail Adapter (LTA) to a nucleic acid molecule comprising the known target nucleotide sequence to produce a ligation product;

(ii) amplifying the ligation product via polymerase chain reaction using a Splint that specifically anneals to the universal LTA, and a first target-specific primer that specifically anneals to the known target nucleotide sequence to produce a first amplification product; (iii) amplifying the first amplification product via polymerase chain reaction using a Splintspecific primer and a second target-specific primer, wherein the second target-specific primer is nested relative to the first target-specific primer to produce a second amplification product; and

(iv) sequencing the second amplification product using a first sequencing primer and a second sequencing primer, wherein the first sequencing primer and the second sequencing primer are complementary to opposite strands of the second amplification product.

141. A method of determining if a subject in need of treatment for cancer will be responsive to a given treatment, the method comprising: detecting, in a tumor sample obtained from the subject, the presence of an oncogene rearrangement according to the method of any one of embodiments 1- 140; wherein the subject is determined to be responsive to a treatment targeting the oncogene rearrangement product if the presence of the oncogene rearrangement is detected.

142. The method of embodiment 141, wherein the oncogene rearrangement is an ALK oncogene rearrangement, and wherein the subject will be responsive to a treatment selected from the group consisting of an ALK inhibitor; crizotinib (PF-02341066); AP26113; LDK378; 3-39; AF802; IPI-504; ASP3026; AP-26113; X-396; GSK-1838705A; CH5424802; andNVP- TAE684.

143. The method of embodiment 141, wherein the oncogene rearrangement is an ROS1 oncogene rearrangement, and wherein the subject will be responsive to a treatment selected from the group consisting of an ALK inhibitor; crizotinib (PF-02341066); AP26113; LDK378; 3-39; AF802; IPI-504; ASP3026; AP-26113; X-396; GSK-1838705A; CH5424802; andNVP- TAE684.

144. The method of embodiment 141, wherein the oncogene rearrangement is an RET oncogene rearrangement, and wherein the subject will be responsive to a treatment selected from the group consisting of a RET inhibitor; DP-2490; DP-3636; SU5416; BAY 43-9006; BAY 73-4506 (regorafenib); ZD6474; NVP-AST487; sorafenib; RPI-1; XL184; vandetanib; sunitinib; imatinib; pazopanib; axitinib; motesanib; gefitinib; and withaferin A.

145. The method of any one of embodiments 141 - 144, wherein the cancer is lung cancer.

146. A method of treating cancer, the method comprising: detecting, in a tumor sample obtained from a subject in need of treatment for cancer, the presence an oncogene rearrangement according to the method of any one of embodiments 1 - 140; and administering a cancer treatment which is effective against tumors comprising the oncogene rearrangement. [0197] Having now generally described the disclosure, the same will be more readily understood through reference to the following examples that are provided by way of illustration, and are not intended to be limiting of the present disclosure, unless specified.

EXAMPLES

Example 1: Detection of FGFR2 gene RNA fusion with known upstream partners using gBlocks as the template.

[0198] RNA fusion detection starts with a reverse transcription kit for the reverse transcription from RNA to cDNA. To test the feasibility, the DNA synthesis template gBlocks (which are double-stranded DNA fragments) containing the fusion exons are used. The FGFR2 gene fusion gBlocks comprise an FGFR2 exonl9-AHCYLl exon5, an FGFR2 exonl9-BICCl exon3, and an FGFR2 exon 19-GAB2 exon2. The gBlocks are mixed with human genomic DNA and then sheared to 300 bp using a DNA fragmentase from Twist Bioscience. Then the fusion detection workflow is followed to detect the fusions.

[0199] The RNA fusion detection is shown using synthetic DNA gBlocks that comprise an exon region. The OFP and IFP designs are based on cDNAs. Both designs comprise a forward design and a reverse design. The forward design is used for an unknown partner which is at the downstream of the known exon, and the reverse design is at the upstream of the known exon. The IFP 3' end has a distance of 5 - 20 nt to the breakpoint. The forward design uses an anti-sense strand as the binding template, and the reverse design uses a sense strand as the binding template. Exemplary primer sequences used for detecting FGFR2 gene fusion is shown in the Table 3.

[0200] The results are shown in Figure 9. Since the design has a 5 plex IFP and an OFP to cover exons 16, 17, 18, 19, and 20, in Figure 9a, all the targeting exons reads were observed. All the gene fusion partners are shown in Figure 9b, Figure 9c, and Figure 9d.

Example 2: Detection of NTRK1 gene RNA fusion with a known downstream partner and its verification in RNA samples.

[0201] In some embodiments, the known exons are in the downstream fusion partner. An example is shown using the NTRK1 gene. Based on the downstream exons, IFPs are designed that can bind to a sense template of NTRK1 with different exons from exon8 to exon!4 for a total 7 pl exes. The IFPs had different gaps to the breakpoint with a length of 9 - 17 nt. The IFPs are designed approximately 5 - 10 nt from the breakpoint. Some primer designs are shifted to avoid primer dimers, and the gap is different consequently. [0202] For approach validation, synthetic gBlocks are used. The NTRK1 gene WT gBlocks comprise 3 different templates covering exon8 to part of exonl6. The NTRK1 fusion gBlocks comprise 4 different templates.

[0203] All fusion gBlocks and WT gBlocks are pooled and mixed with equal ratio as one template pool. All WT gBlocks are pooled together as another template pool. Different libraries are constructed from each template pool using the LTA approach. The RNA template ordered from SeraCare is reverse-transcribed into cDNA and was used as the template for the library preparation. Exemplary primer sequences used for detecting NTRK1 gene fusion is shown in the Table 4.

[0204] All unknown partners of the NTRK1 fusion genes are found and shown in Figures 10a to Figures 10c.

Example 3: Detection of ALK intron 19 DNA fusion.

[0205] In some embodiments, the LTA approach is used for the DNA fusion detection. For the DNA fusion, the breakpoint was usually in the intron region so that covering the intron region is necessary to detect unknown DNA fusions (Figure 11). In some embodiments, gene 1 exon 2 was used as one known fusion partner, whereas the unknown exon is in the downstream of a known fusion partner, multiple IFPs needed to tile the intron 2 sequence of gene 1, and the binding target sequence is in the reverse strand of intron 2. In some embodiment, gene 1 exon 2 is used as a known fusion partner, whereas the unknown exon is in the upstream of the known fusion partner, and multiple IFPs are needed to tile intron 1 of gene 1, and the binding target sequence is in the forward strand of intron 1.

[0206] Multiple IFPs are designed for tiling the whole intron region. One IFP 3' end had a 0- 100 nt gap to the next IFP 5' end of the template binding site (excluding adapter region). The tiling design is for NGS sequencing to cover as much as possible breakpoint using shorter amplicon since the NGS read length is below 600 bp. Exemplary primer sequences used for detecting ALK gene fusion is shown in the Table 5.

[0207] The ALK gene has multiple fusion types, most of which appear in exon 20. In some embodiments, the exon 20 of ALK is the known partner in the downstream of the fusion, such as the EML4 exon 6 - ALK exon 20 fusion. A set ofl4-plex IFPs and OFPs are designed to tile all the intron 19 region of the ALK gene to find the unknown upstream fusion partner. The ALK design is tested on the H2228 cell line DNA since it has an EML4-ALK fusion. The results are shown in Figure 12. The fusion reads contain the EML4 intron 6 sequence and the ALK intron 19 sequence, which are referred to the fusion type as EML4 exon 6 and ALK exon 20, respectively. Example 4: Detection of EML4-ALK DNA fusion via ABDA enrichment.

[0208] The EML4-ALK fusion breakpoint is in the intron!9 region of the ALK gene. After NGS sequencing using the LTA approach, the breakpoint is found to be close to inner primer 13 with a 10 nt gap between the 3' end of the IFP13 and the breakpoint. Based on BDA design rules, a Blocker is designed to cover the breakpoint with a 6 nt overlap to the IFP13. For EML4-ALK fusion sample H2228, the Blocker has a 6 nt region that cannot bind to the fusion template, and it can bind to the WT template.

[0209] NGS results are shown in Table 1. The fusion rate is calculated as the fusion reads divided by the sum of the fusion reads and the WT reads. The Fusion Variant Allele Frequency (VAF) is defined as the fusion rate of H2228 sample without BDA enrichment. The Fusion Variant Reads Frequency (VRF) is defined as the fusion rate of H2228 sample after BDA enrichment. The enrichment fold is calculated based on Table 1 equation (4). The enrichment fold for H2228 EML4-ALK fusion is approximately 7 folds.

[0210] Table 1: ABDA enrichment fold calculation for fusion variants. We use two samples to test the ABDA approach: NA18537 as the WT sample and H2228 as the fusion variant sample. Without the ABDA enrichment, the fusion Variant Allele Frequency (VAF) is 31% for H2228 fusion samples. After ABDA enrichment, the Variant Reads Frequency (VRF) is 75.5%. The enrichment fold calculation follows equation (4) and results in the value of 7 folds.

Table 1. ABDA enrichment fold calculation for fusion variants

Fusion Rate = Fusion Reads/(Fusion Reads + WT Reads)

VAF = Fusion Rate of H2228 noB

VRF = Fusion rate of H2228 wB

Enrichment Fold = ((1-VAF)/VAF)/((1-VRF)/VRF) = 7 [0211] Table 2: Exemplary sequences for adapter. We design two exemplary adapters, one has a length of 13 bp in the DSE region which comprises the LTA-UMI-up and the LTA-UMI- bottom (FLTA-UMI-up and FLTA-UMI-bottom, respectively). Another one has a longer length of 22 bp in the DSE region which comprises the LTA-UMI-long-up and the LTA-UMI-bottom (FLTA-UMI-long-up and FLTA-UMI-long-bottom, respectively). The UMI sequences are shown as HHHHHHHHHHHHHHH " in the sequences. The three UP sequences are named FusTaiSeqAdaPrimerl, FusTaiSeqAdaPrimer2, FusTaiSeqAdaPrimer3.

Table 2. Examples of adapter sequences.

[0212] Table 3: Exemplary primer sequences for FGFR2 fusion detection. Table 3 shows the primer sequences for FGFR2 gene fusions which is targeting all the exon regions and is designed based on FGFR2 as the upstream fusion partner. This table includes outer primers, inner primers, inner primers with sequencing adapters and Blocker sequences for the ABDA enrichment.

Table 3. Examples of primer sequences for FGFR2 fusion detection.

FGFR2exl AD iFP-9 TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGctctgcatggttgacagtc

[0213] Table 4: Exemplary primer sequences for NTRK1 fusion detection. Table 4 shows the primer sequences for targeting the NTRK1 gene fusion which targets NTRK1 as a downstream partner. This table includes 7 inner and outer primer designs and a primer with sequencing adapters for targeting 7 exons from exon 8 to exon 14. Table 4. Examples of primer sequences for NTRK1 fusion detection

[0214] Table 5: Exemplary primer sequences for ALK fusion detection. Table 5 shows the 14 pl ex primer sequences for targeting ALK intron 19 and one ABDA design based on H2228 DNA fusion breakpoint. The ABDA design includes the OFP, the IFP and the IFP with sequencing adapter and Blocker sequences. Table 5. Examples of primer sequences for ALK fusion detection