REFERENCE TO SEQUENCE LISTING, TABLE OR COMPUTER PROGRAM The Sequence Listing is concurrently submitted herewith with the specification as an

ASCII formatted text file via EFS-Web with a file name of Sequence Listing.txt with a creation date of February 23, 2016, and a size of 5.11 kilobytes. The Sequence Listing filed via EFS-Web is part of the specification and is hereby incorporated in its entirety by reference herein.

Multiplex-PCR consists of multiple primer sets within a single PGR mixture to produce amplicons that are specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test ran that would otherwise require several times of the reagen ts and more time to perform. Annealing temperature for each of the primer sets must be optimized to work correctly within a single reaction.

Commercial kits for multiplexing PGR general reagents are available. The technique of multiplex PGR has been used for target enrichment for next-generation sequencing (NGS), which refers to high throughput parallel DNA sequencing technologies. Millions or billions of DNA strands can be sequenced concurrently, yielding substantially high throughput.

One major reason for amplicon drop-out is preferential amplification of the short overlapping regions between two overlapping amplicons during amplification. Currently, to amplify two overlapping DNA amplicons, the primer pairs specifically targeting each amplicon are physically separated into different reaction wells, tubes or micro-droplets. For example, BRCA1 and BRCA2 genes contain large exons that require PGR amplification of overlapping DNA amplicons to ensure 100% base coverage. Ion AmpliSeq BRCAl and BRCA2 Panel, Qiagen GeneRead Human BRCAl and BRCA2 Panel, and Multiplicom

BRCA MASTR Dx separate primer pairs into 3, 4, 5 primers pools, respectively, primarily due to the inability to amplify overlapping amplicons efficiently. However, multiple primer pools significantly complicate the workflow and increase the cost of testing. RainDance Technologies overcomes this issue by separating PGR primers into thousands of micro- droplets, but on a special expensive instrument.

Combining all PGR primers for two overlapping DNA regions in one multiplex reaction produces four products resulting from four different combinations of the two forward primers with the two different reverse primers. FIG. 1 shows a conventional PGR method. The four PGR products (FIG. 1) are two targeted amplicons (AmpHcon 1 and Amplicon 2), one long amplicon (Amplicon 4 long) spanning the entire region of the two targeted amplicons and one short amplicon (Amplicon 3_overlap) containing only the overlapped regions between the two targeted amplicons. Using conventional primer design and PGR conditions, during cycling, the longest amplicon (Amplicon 4_long) serves as DNA template for all four amplicons' amplification, and each of the two targeted amplicons

(Amplicon 1 & 2) serves as DNA template for amplification of its own amplicon as well as the shortest amplicon (Amplicon 3_overlap). Assuming that all amplifications occur at 100% efficiency, at PGR cycle n, the amount of the four products - Amplicon 1, Amplicon 2, Amplicon 4_long and Amplicon 3_overlap - will be n x 2", n x 2", 2", and n ² x 2", respectively. The amount of shortest amplicon (Amplicon 3 overlap) is n times higher than that of each of the two targeted amplicons (Amplicon 1 & 2) which in turn is n times higher than the amount of the longest amplicon (Amplicon 4 Jong).

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 illustrates a traditional multiplex PGR (prior art), for amplification of overlapping target segments.

FIG. 2 illustrates one embodiment of a first round of PGR of the present invention, which is the amplification by target-specific primers. Fl and Rl are the forward and reverse primers of target segment 1 , while F2 and R2 are the forward and reverse primers of target segment 2. F2 ^A is a partial sequence of the 5 '-end portion of the F2 primer. Tag oligomers of tl , t2 and t3 do not bind to the target. Tags t2 and t3 may share the same sequences and tl is different. 1, 2, 3 and 4 indicate the amplification products from the combination of four primers. Amplification of Amplicon 3, the short products from F2 and RL is inhibited, by the formation of a stem-loop structure.

FIG. 3 shows the sizes and locations of Amplicons 1-4 on human chromosome 13 for Examples 1 and 2,

FIG. 4 shows the agarose gel electrophoresis results of the amplification products after the first round of PGR (Examples 1 and 2),

FIG. 5 is a schematic illustration of the gene-specific amplicons used in examples 3-6. CDS: protein coding sequence region of a gene; ROI: region of interest. In this example, ROI includes the CDS of BRCA2 gene exon 27 plus 20 bp upstream and 20 bp downstream of exon 27 (CDS ± 20 bp).

FIG. 6 shows the agarose gel electrophoresis results of amplification products after the first round (Example 3) and second round of PGR (Example 4).

FIG. 7 is a chart showing the coverage data from NGS results of amplicons (Example 6).

An "amplicon" is a piece of DNA or RNA that is the source and/or product of natural or artificial amplification or replication events. In this context, "amplification" refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as PGR product.

"Locked nucleic acids" (LNA™) are a class of high-affinity RNA analogues in which the ribose ring is "locked" by a methylene bridge connecting the 2'-0 atom and the 4'~C atom resulting in the ideal conformation for Watson-Crick binding. This modification significantly increases the melting temperature of an oligonucleotide and is also very nuclease resistant (www.exiqon.com/lna4echiiologv).

"Peptide nucleic acids" (PNAs) are synthetic homologs of nucleic acids in which the phosphate-sugar polynucleotide backbone is replaced by a flexible pseudo-peptide polymer to which the nucleobases are linked. Because the PNA strand is uncharged, a PNA-DNA duplex will have a higher melting temperature than the corresponding DNA-DNA duplex.

"Universal (PGR) primers" are non-target specific primers that hybridize to universal tags (non-target specific tags such as e.g. tl, t2 and/or t3 in FIG. 2) flanking both ends of any DNA insert sequences. PGR that uses universal primers can amplify any DNA inserts that are flanked by their complementary tag sequences.

The inventors have discovered a scalable multiplex PGR technology, SLIMAMP™ (Stem-Loop Inhibition-Mediated Amplification), which allows for parallel amplification of hundreds of thousands of amplicons in one tube. This novel multiplex PGR method can simultaneously amplify overlapping amplicons without the drawbacks of conventional multiplex PGR, which predominantly amplifies the short overlapping nucleic acid sequences. The present method is a target enrichment method, which enriches copies of target amplicons over copies of the overlapping regions of amplicons.

The present invention is directed to a method for selectively amplifying target nucleic acid fragments having an overlapping region, without a predominant amplification of the short overlapping nucleic acid sequences. The present invention allows all primers in a single primer pool without introducing any additional expensive equipment.

The present method comprises the steps of: (a) obtaining a first nucleic acid sequence comprising a first tag t2 and a first forward primer Fl, (t2Fl), complementary to a first target nucleic acid fragment (amplicon 1), (b) obtaining a second nucleic acid sequence comprising a second tag tl and a first reverse primer Rl , (tlRl ), complementary to the first target nucleic acid fragment (amplicon 1), (c) obtaining a third nucleic acid sequence comprising the second tag tl and a second forward primer F2, (tlF2), complementary to a second target nucleic acid fragment (amplicon 2), (d) obtaining a fourth nucleic acid sequence comprising a third tag t3 and a second reverse primer R2, (t3R2), complementary to the second nucleic acid fragment (amplicon 2), wherein the first and the second target nucleic acid fragments have an overlapping region, (e) mixing the first and the second target nucleic acid fragments, the first, the second, the third, and the fourth nucleic acid sequences, and an effective amount of reagents necessary for performing a polymerase chain reaction (PGR); (f) cycling the mixture of (e) throug denaturing, annealing and primer extension steps of PGR for at least two times, and (g) cycling the mixture of (f) through denaturing, annealing and primer extension steps of PGR at an annealing temperature higher than that in step (f) to obtain amplification products. The above method steps describe a first round of PGR cycles.

Fl, Rl, F2, R2 are gene-specific primers, which are complementary to specific regions of genomic DNA. The length of these primers can be chosen by a person skilled in the art. In general, the gene-specific primers are 6-40, 10-50, 10-40, 10-100, 20-40, or 20-50 nucleotides in length.

Tags tl and t2 are two different universal tag sequences. Tag t3 can have the same or different sequence as t2. Tag oligomers of tl, t2 and t3 do not bind to the target sequences. Each tag is at the 5 'end of each gene-specific primer.

The present invention is illustrated in FIG. 2. FIG. 2 is for illustration purposes and is not meant to limit the invention to the drawings only. The arrangement of tags, forward primers, reverse primers, target nucleic acids, amplicons as described in steps (a)-(d) is shown at the upper pari of FIG. 2. FIG. 2 illustrates one embodiment of the present invention, in which F2 ^A is a partial sequence of the 5 '-end portion of the F2 primer. In other embodiments of the invention, F2 ^A is replaced with 0 nucleotide (not present) or replaced with F2 (full sequence of F2 primer) in FIG . 2.

In step (f), the mixture of nucleic acids and reagents go through the PGR cycles of denaturing, annealing and primer extension steps at least two times, such as 2-5 times or 2-10 times, at standard PGR temperatures or conditions known to a person skilled in the art. In the very first PGR cycle, amplicons tagged only at one end are generated. In the second PGR cycle, the one-ended tagged amplicons then serve as templates for the other tagged primers to generate 2-ended tagged amplicons. Step (f) is illustrated at the middle part of FIG. 2, wherein Amplicon 1 (Fl+Rl), Amplicon 2 (F2+R2), Amplicon 4_long (F1+R2), and Amplicon 3 overlap (F2+R1) are generated after the initial cycles. After at least two cycles of PGR, complete amplicons with the tag sequences at both ends are generated and ready for the next round of PGR cycles with an increased annealing temperature of step (g).

In step (g), the mixture of (f) goes through more cycles of PGR of denaturing, annealing and primer extension; this time at an annealing temperature higher than that in step (f), which is an important feature of the present invention. The annealing temperature is increased to prevent the shortest amplicon (Amplicon 3_overlap) to be amplified

predominantly.

If the annealing temperature is not increased (as in a conventional PGR method), all amplification follows a conventional way where Amplicons 1 , 2, and 4 long can all serve as templates for the amplification of the short amplicon (Amplicon 3_overlap); this is because Amplicons 1, 2, and 4_long all contain the gene-specific parts of the forward and reverse primers of Amplicon 3 overlap. As a result of the amplification (without increasing annealing temperature), the amount of the shortest amplicon (Amplicon 3_overlap) is n ² 2 ⁿ , which is n times higher than that of each of the two targeted amplicons (Amplicons 1 & 2, n x 2 ^B ), assuming all amplicons have the same amplification efficiencies, In practice, it is observed that amplification efficiency is affected by amplicon length in which shorter amplicons correlate with higher amplification efficiency (Mallona I, et al, BMC

Bioinformatics 201 1, 12:404), and therefore, the shorter amplicon amplifies even more favorably. In step (g) of the present invention, the annealing temperature is increased, and thus a successful primer-template hybridization requires not only the gene-specific parts of the primers, but also the tag parts. When the annealing temperature is increased, Ampiicons 1, 2, and 4 long can no longer serve as templates for the amplification of the short amplicon (Amplicon 3_overlap), and only the short amplicon itself (Amplicon 3 overlap) can serve as a template for itself. The present invention features (i) an increasing annealing temperature after the initial at least two PGR cycles, and (ii) a proper arrangement of tags (tl, t2, t3) associated with each amplicon, in particular, F2 and Rl primers are tagged with the same tag tl; such features result in production of 2 ⁿ copies of all ampiicons in theory, because each amplicon can only use its own amplicon as a template for its amplification. This is already an impro vement over a conventional method, in which Amplicon 3 (short overlap) would yield n times higher than that of target ampiicons. In practice, shorter ampiicons typically amplify more efficiently than longer ampiicons.

In step (g), the annealing temperature is at least 2°C higher than the annealing temperature in step (f). For example, the annealing temperature is about 2-35°C, 4-35°C, 5- 25°C, 6-20°C, 6-15°C higher than the gene-specific annealing temperature in step (f). In step (g), the PGR cycling is repeated at least 2 times, e.g., 2-50 times, preferably 2-5, 5-10, 10-30, 10-40, or 10-50 times.

Any added bases can increase the primer's melting temperature when the primer sequences match the template 100%. In the present invention, the tag sequences are at least 2 or 3 nucleotides in length, and can be 5-100, 3-40, 10-30, 10-40, 10-50 nucleotides long. Preferably, tags are designed to add at least 5°C (e.g., 5-10°C or 5-15°C) to the melting temperature of the gene-specific untagged primers. The tag sequences provide a higher annealing temperature of the cycling conditions after the initial minimal 2 cycles in PGR . Tag sequences can be modified or unmodified nucleic acids. Some modified bases (e.g. LNA or PNA) have higher annealing temperatures than their corresponding natural bases. When shorter tag sequences are desired for various reasons, those modified bases can be used instead of the natural bases.

In one embodiment of the invention, the Tm (melting temperature) of tag tl sequences on both ends of amplicon 3 is high enough to form a tight tl-stem, which prevents the hybridization of primer tlF2 to the amplicon 3 template, and inhibits a further exponential amplification of amplicon 3. In order to inhibit the binding of primer tlF2 to amplicon 3 template, the Tm of the tl-stem at the end of amplicon 3 should be the same or higher than the Tm of tlF2 oligo alone. The melting and hybridization of tl-stem at the ends of amplicon 3 follow intramolecular kinetics, and are more favorable than those of the regular intermolecular oligo duplex reactions. Therefore, the same two short complimentary oligo sequences (e.g. 2 to 100 nucleotides) have a much higher Tm when they form a stem connected by a non-complimentary loop in one molecule than Tm of the same two complimentary oligo sequences forming a linear duplex. In addition, the Tm of the stem is not only influenced by the stem sequences but also the loop length (corresponding to the overlapping region). In comparison to a small loop size (e.g. 2-200, 5-200, or 10-100 nucleotides), a large loop size (e.g. greater than 500 nucleotides such as 500-1000 or 500- 1500 nucleotides) reduces the stem hybridization rates, possibly due to the decreased probabilities of contact between the ends of the larger loop, and resembles the kinetics of regular intermolecular DNA duplex formations.

In one embodiment, the second nucleic acid sequence optionally further comprises a full sequence (F2), or a partial sequence (F2 ^A ) consecutively from the 5 'end of the second forward primer (F2), in between the second tag (tl) and the first reverse primer (Rl). In FIG. 2, this optional embodiment of primer tl F2 ^A Rl is shown. Dependent on the length of F2, in one embodiment, the partial sequence of F2 ^A is 1-50, 1-20, 1-10, or 1-5 nucleotides shorter than F2. For example, the partial sequence of F2 ^A may be 2-40, 4-40, 8-40, 8-30, or 8-20 nucleotides. In another embodiment, the partial sequence of F2 ^A contains 10-50, 10-90, 20-80, 30-70, 40-90, or 50-90% of the F2 sequence.

As shown at the lower part of FIG. 2, after step (g), amplicon 1 (Fl+Rl), Amplicon 2 (F2+R2), and Amplicon 4_long (F1+R2) are amplified exponentially by PGR, while the amplification of Amplicon 3 overlap (F2+R1) is inhibited. This is because F2 and Rl primers are tagged with the same tag tl, and therefore in the presence of F2 ^A with a proper length, a strong stem loop structure containing the sequences of tl and F2 ^A forms and prevents the hybridization of primer tlF2 to the amplicon 3 template, which inhibits the further exponential amplification of amplicon 3. The proper length of F2 ^A that enables a strong stem loop structure may var depending on the Tm of tag tl. FIG. 2 illustrates the stem loop formation with tl and F2 ^A , which is a preferred embodiment. In another embodiment, as described above, the Tm of tag tl sequences on both ends of amplicon 3 are high enough to form a tight tl-stem, and the presence of F2 ^A is not required.

In another embodiment of the invention, the above amplification products after the first round of PGR as described above are amplified further by a second round of PCR amplification with universal primers that bind to tl and t2 in the first round. In this embodiment, t3 is required to be the same as t2 during the first round of PGR. The second PGR round comprises the steps of: (h) mixing the amplification products from (g), either treated or untreated, with a first and a second universal PGR primers that bind to tl and t2 respectively, and an effective amount of reagents necessary for performing a PGR, wherein both universal PGR primers do not comprise sequences complementar to the first and the second target-specific nucleic acid sequences; and (i) cycling the mixture of (h) through denaturing, annealing and primer extension steps of PGR at standard PGR temperatures and conditions known to a person skilled in the art to obtain second amplification products.

In step (i), the PGR cycling is repeated at least 2 times, e.g., 5-50 times, preferably 2- 5, 5-10, 10-30, 10-40, or 10-50 times.

During the second round of PGR, the amplifications of the short overlapping

Amplicon 3_overlap and the long AmpHcon 4__long are further inhibited because their primer binding sites are blocked by the strong stem formation of tl at both ends of the Amplicon 3_short and t2 at both ends of the Amplicon 4__long. Therefore, the amplifications of the first and the second target nucleic acid amplicons (Amplicons 1 and 2) dominate in the second round of PGR. The final products can be used for different purposes, suc as next generation sequencing (NGS).

In the second round of PGR, the product of (g) from the first round of PGR is optionally pre-treated before the step (i), for example, by dilution, single-strand exonuclease digestion, purification, or adaptor ligation.

The following examples further illustrate the present invention. These examples are intended merely to be illustrative of the present inv ention and are not to be construed as being limiting.

EXAMPLES

Table 1 and FIG, 3 show the sizes and locations of Amplicons 1-4 on human genome hgl9. Amplicon 3 is the overlap between Amplicons 1 and 2, Amplicon 4 is the long amplicon cov ering the sequences of both Amplicons 1 and 2.

Table 2 shows oligonucleotide sequences used in Examples 1 and 2, and FIG. 4 SEQ ID NOs: 1-4 are gene specific primers for BRCA2 ampiicon 1 and ampiicon 2 without tag sequences. SEQ ID NOs: 5-6 are tag sequences from Illumina TSCA tags. SEQ ID NOs: 7-16 are the tagged primers used in the example experiments.

SEQ ID NO: 7 was used in standard multiplex PGR only (FIG. 4, Lane 3). SEQ ID NOs: 8-14 were used in the present invention (Lane 4-8). SEQ ID NOs: 15-16 were used in both the standard multiplex PGR and the present invention (Lane 3-8).

Tm are evaluated by Oligo Analyzer 3.1 (IDTDNA) with 1,5 mM of Mg ⁺² concentration.

ces,

Table 3 shows primer mix information in Examples 1 and 2.

F2_R1 Short AmpEicort

Loop Length (nt); Stem

AsTsplicon2 Gliges; Stem length (nt)

1: lOObp Ladder

Smgleplex Control

Not applicable mix'

STD _„ u!tipiex _„ ctrl tl Fl 12 Rl tl F2 t2 R2 no stem loop

Stem tl-F2 ^A 0 tl Fl tl Rl tl F2 t2 R2 195; tl_only; 20

Stem tl- F2 ^A 4 t2 Fl tl F2 Rl tl F2 t2 R2 195; tl + 4nt of F2; 24

Stem tl-F2 ^A 8 t2 Fl tl F2 ^A 8 Rl tl F2 t2 R2 195; tl + 8nt of F2; 28

Stem tl-F2 ^A 12 t2 Fl tl F2 ^A 12 Rl tl F2 t2 R2 195; tl + 12nt of F2; 32

Stem tl-F2 ^A 16 t2 Fl tl F2 ^A 16 Rl tl F2 t2 R2 195; tl + 16nt of F2; 36

*Contains each singleplex product from separate amplifications mixed at the equal volume ratio

A representative PGR mixture of 25 uL included the following components:

12.5 uL of 2x Multiplex Master Mix (KAPA Biosystems, Cat# K5802), 2 μΕ human genomic DNA (Promega, Cat#G3041) diluted to 5 ng/uX in low TE buffer (USB, Cat#75793), 6.5 μΕ nuclease-free water, and 4 μΕ of gene-specific primer mix (1.25 μΜ each, see Multiplex Primer Mix Lanes 3-8 in Table 3).

The singleplex (FIG. 4, Lane 2) PGR, the standard multiplex (FIG. 4, Lane 3) PGR, and the stem-forming multiplex (FIG. 4, Lanes 4-8) PGR were all performed on a thermal cycler as follows:

1 cycle 95°C 2 minutes Enzyme activation and initial DNA denaturation

5 cycles 95°C 30 seconds

60°C 90 seconds Annealing/extension

95°C 30 seconds Denaturation

72°C 90 seconds Annealing/extension at an increased temperature

1 cycle 72°C 5 minutes Final extension 1 cycle 8°C Hold

Example It Agarose Gel Electrophoresis

The products from example 1 were analyzed on an E-base device (Life Technologies). Two \iL of the product was diluted to a final volume of 20 uL with nuclease-free water and loaded onto a 2% SizeSelect E-gel. DNA electrophores s of diluted PGR products (Lanes 2-8) and 1 Kb Plus DNA ladder (Invitrogen, Cat# 10488-090, Lane 1) was performed, and at the end of the run, a digital image of the gel was captured by an E-gel Imager (Life Technologies). Results are shown in FIG. 4.

In Lane 2 of FIG. 4, equal amounts of the products of singleplex reactions from Exampl 1 are mixed and can be seen together on the gel, designated Amplicons I and 2. Standard multiplex PGR predominantly produces Amplicon 3 (Lane 3), which is PGR product amplified from the overlap of Amplicons 1 and 2. Amplicon 4, the entire region covered by Amplicons 1 and 2, ca faintly be seen in Lane 3. Lanes 4 and 5, which contain stem oligonucleotides of tl only and tl plus partial F2 (4 nucleotides from the 5 'end), respectively, show similar patterns with three detectable bands: Amplicons 1, 2 and 3. In Lanes 6-8, all Amplicons 1 , 2, and 3 can be seen, but amplicon 3 has decreased substantially relative to Lanes 3-5.

Table 4 and FIG. 5 show the amplicon sizes and locations on human genome hgl9 targeting BRCA2 Exon 27 (B2X27) used in examples 3-6.

Table 5 shows oligonucleotide sequences used in Examples 3, 4, 5, and 6 for BRCA2 (B2) Exon 27 (X27) amplification.

*Lower case indicates tag sequences; Underline indicates inserted partial forward primer sequences from the next amplicon; un-iabeled, upper case sequences are gene-specific sequences.

Example 3: First Round of Gene-specific PCR Amplification for BRCA2 exon 27 (B2X27) A representative PCR mixture of 25 _, uL included the following components:

12.5 uL of 2x Multiplex Master Mix (KAPA Biosystems, Cat#KK5802), 6 uL of DNA (Coriell, Cat#NA 19240 or NA 14622) diluted to 5 ng uL in low TE buffer (IDT, Cat#l 1-05-01-09), 2.5 uL nuclease- free water, and 4 μ£ of gene-specific primer mix (1.25 μΜ each).

The conventional primer mix contained the following six oligos from Table 5: SEQ ID

NOs: 17 and 18 (B2X27 amplicon 1), 19 and 20 (B2X27 amplicon 2), and 21 and 22 (B2X27 amplicon 3). The SLIMAMP™ primer mix (the present invention) contains the following six oligos: 17 and 23 (B2X27 amplicon 1), 24 and 25 (B2X27 amplicon 2), and 21 and 22 (B2X27 amplicon 3).

The standard multiplex and stem- forming multiplex PCR is performed on a thermal cycler as follows: 1 cycle minutes Enzyme activation and initial DNA denaturation

5 cycles 95 °C 15 seconds Denaturation

60°C 6 minutes Annealing/extension

25 cycles 95°C 30 seconds Denaturation

72°C 3 minutes Annealing/extension at increased temperature

1 ³ C Hold

The gene-specific products in Example 3 were diluted 1000-fold. A representative PGR mixture of 25 \iL included the following components: 2.5 μΕ ΙΟχ reaction buffer, 0.5 \iL dNTPs, 0.25 ,uL enzyme (Roche, Cat#12140314001), 2 μΕ of the diluted product from Example 3, 2 xL of Illumina Index Forward primer, 2 μΕ Iliumina Index Reverse primer (25 μΜ primer stock from TruSeq Custom Amplicon Index Kit, Cat#FC- 130- 1003), and 15.75 iL nuclease-free water.

PCR amplification was performed as follows:

1 cycle 95°C 4 minutes Initial DNA denaturation

95°C 30 seconds Denaturation

20 cvcles 66°C 30 seconds Annealing

72°C 60 seconds Extension cycle 5 minutes Final extension

cycle 8°C Hold

The product was purified by adding 18 μΕ of Agencourt AMPure XP beads (Beckman Coulter, Cat#A63881), separating the beads from the supernatant, and discarding the

supernatant. Two washes of 70% ethanol were used to wash the beads, a d the product was eluted from the beads using 32 μΕ nuclease-free water. The concentration of the product was then quantified using 2 μΕ of the product diluted in 198 uL of Qubit High Sensitivity buffer (Invitrogen, Cat#Q32854).

Example 5: Agarose Gel Electrophoresis

The products from example 3 and example 4 were analyzed on an E-base device (Life

Technologies). Two ,uL of the product was diluted to a final volume of 20 μΐ. with nuclease- free water and loaded onto a 2% SizeSelect E-gel. DNA electrophoresis of diluted PCR products and 50 bp DNA ladder (Invitrogen, Cat# 10488-043) was performed. At the end of the run, a digital image of the gel was captured by an E-gel Image (Life Technologies). Results are shown in FIG. 6.

The products from example 3 (First round of PCR products) are shown in Lane 1-4 of FIG. 6. Lanes 1 and 2 demonstrate the products of conventional multiplex PCR. The small amplicons, which are the overlaps of amplicons 1 with 2 and 2 with 3, were produced along with amplicons 1-3. On the other hand, SLIMAMP™ reactions, loaded in Lanes 3 and 4, produce mainly amplicons 1-3, with very little of the overlapping amplicons.

The products from Example 4 (second round of PCR products) are shown in Lanes 5-8 of FIG. 6, which are products after universal PCR and cleaned up by beads. In Example 4 (Lanes 5-8), amplicons were tagged and amplified further, demonstrated by the increase in nucleotide size when compared with Lanes 1-4. The samples that originally underwent conventional multiplex PCR produced primarily the undesired short overlapping amplicons (Lanes 5-6), while the SLIMAMP™ samples (the present invention) contain only the targeted amplicons 1-3 (Lanes 7-8).

Example 6: NGS Library Normalization and Sequencing

Each product from Example 4 was normalized to 4 mM using 10 mM Tris-HCl w/ 0.1%

Tween 20 (Teknova, Cat#T7724). All normalized products from Example 4 are mixed in equal volume (3 μΕ each) to create a library mix. 5 ah of the library mix is added to 5 uL of 0.2 N NaOH to denature the library. A 20 pM library is prepared using HT1 buffer, loaded, and sequenced with a 250 bp paired-end read length (Illumina, Cat#MS- 102-2003). The resulting Fastq sequencing reads for each sample was then aligned to the hg!9 reference genome by BWA-MEM. The paired-end reads were then merged and the coverages for each amplicon regions were analyzed, The coverage infomiation is shown in FIG. 7,

FIG. 7 depicts the percentage of NGS reads attributed to each amplicon in each sample. In the conventional multiplex PCR, the overlapping aniplicons were amplified more efficiently than the target amplicons and count for 78% of the total NGS reads, due to their much smaller sizes (60 and 82 bp). In the SLIMAMP™ samples, all the undesired overlapping amplicons were inhibited with close to 0% detected in NGS. With respect to the targeted amplicons in both the conventional and SLIMAMP™ samples, as expected, amplicon 1 (267bp) had higher percentage than both amplicons 2 (324bp) and 3 (348bp) because a smaller size (amplicon 1) typically amplifies more efficiently.

It is to be understood that the foregoing describes preferred embodiments of the present invention and that modifications may be made therein without departing from the scope of the present invention as set forth in the claims.