FORMALIN FIXED PARAFFIN EMBEDDED (FFPE) CONTROL REAGENTS

[0001] This application claims priority to U.S. Provisional Patent Application No. 62/232,261, filed September 24, 2016, which is incorporated herein by reference in its entirely.

BACKGROUND

[0002] A significant challenge facing testing laboratories is quality control. Some reports have indicated that mutations in cancer genes were correctly identified by only 70% of testing laboratories (Bellon, et al. External Quality Assessment for KRAS Testing Is Needed: Setup of a European Program and Report of the First Joined Regional Quality Assessment Rounds. Oncologist. 2011 April; 16(4): 467-478). Questions have been raised regarding how to monitor next generation sequencing and assays as well as the concordance of variant calls across multiple platforms, library preparation methods, and bioinformatic pipelines. Compositions and methods providing a flexible, single reagent representing specific genetic variants are desired by those of ordinary skill in the art and are described herein.

SUMMARY

[0003] The disclosure provides compositions, controls, plasmids, cells, methods and kits comprising nucleic acid molecules.

[0004] In one embodiment, a nucleic acid molecule comprising multiple variants of a reference is disclosed. In other embodiments, a mixture or combination of nucleic acid molecules comprising variants of the reference sequence are disclosed.

[0005] In certain embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprise one or more variants present at a high or low-frequency.

[0006] In certain embodiments, the disclosure provides a control reagent comprising multiple nucleic acid molecules.

[0007] In yet another embodiment, a kit comprising at least one nucleic acid molecule or mixture of nucleic acid molecules comprising variants is disclosed

[0008] In another embodiment, a method for confirming the validity of a sequencing reaction is disclosed. The method comprises including a known number of representative sequences and / or variants thereof in a mixture comprising a test sample potentially comprising a test nucleic acid sequence, and sequencing the nucleic acids in the mixture, wherein detection of all of the representative sequences and / or variants in the mixture indicates the sequencing reaction was accurate.

[0009] The disclosure also provides a composition comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage.

[0010] In certain embodiments, a method is provided that comprises sequencing a nucleic acid species in order to calibrate a sequencing instrument.

[0011] In yet other embodiments, the disclosure provides plasmids and cells encoding the nucleic acids or mixture of nucleic acids disclosed herein.

[0012] The disclosure also provides a plasmid and/or a cell comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a

predetermined percentage.

[0013] The disclosure further provides a frequency ladder. The frequency ladder comprises a plurality of variants at different frequencies.

[0014] The disclosure also provides a method of for preparing a formalin fixed paraffin-embedded (FFPE) control, the method comprising: a) obtaining a defined concentration of cellular material; b) introducing in to the cellular material a nucleic acid molecule or mixture of nucleic acid molecules comprising multiple variants of a reference sequence or a mixture of variants with the reference sequence; c) mixing the cellular material of b) with a gelling polymer, creating a gel/cellular material; and d) adding the gel/cellular material to a mold with a defined shape until the gelling polymer solidifies.

[0015] In certain embodiments, the method is carried out with a mixture of variants, wherein the variants comprise at least one single nucleotide polymorphism (SNP), multiple nucleotide polymorphisms (MNP), insertion, deletion, copy number variation, gene fusion, duplication, inversion, repeat polymorphism, homopolymer of a reference sequence, and / or a non-human sequence.

[0016] In yet other embodiments, the method is carried out with a nucleic acid molecule or mixture of nucleic acid molecules comprising multiple variants comprises at least 30 variants. In other embodiments, the nucleic acid molecule or mixture of nucleic acid molecules used in the methods comprises a variant is related to cancer, an inherited disease, infectious disease.

[0017] The disclosure also provides for a kit comprising a formalin fixed paraffin-embedded (FFPE) control produced by the method of the invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 provides exemplary EGFR amplicon selection.

[0019] Figure 2 is a graph showing variant frequency at each nucleotide position as well as percentage A and G content. Sequences 1-5 are the same and are used to dilute out sequences 6-10. Each sequence is found in its own cassette, and all cassettes are found in the same plasmid. This design provides an absolute truth - e.g., there is 10% sequence 6 in this design. In contrast to mixing with genomic sequence, this provides the most precision when making a 10% mix. This could be used to calibrate assays.

[0020] Figure 3 is a schematic of an exemplary plasmid with 10 sequences and restriction sites, leading to equal ratios of each sequence.

[0021] Figure 4 is a graph showing the frequency percentage per run comprising Panel A (FLT3, PDGFRA, FGFR3, CSF1R, EGFR, HRAS, and TP53).

[0022] Figure 5 is a graph showing the frequency percentage per run of Panel A and Panel B (TP53, PIK3CA, GNA11, VHL, FBXW7, RET, HNF1A, and STK11)

[0023] Figure 6 is a graph showing the frequency percentage per run of Panel A, Panel B, and Panel C (RBI, EGFR, ABL1, ERBB2, and ATM).

[0024] Figure 7 is a graph showing the frequency percentage per run of Panel A, Panel B, Panel C, and Line D, which represents the number of reads (i.e., coverage)

[0025] Figure 8 is a graph showing the number of variants (deletions, insertions, complex, multiple nucleotide variants (MNV), and single nucleotide variants (SNV)) and average number of variants detected across multiple sites using CHPv2 (AMPLISEQ™ Cancer Hotspot Panel version 2), TSACP(TRUSEQ™ Amplicon Cancer Panel), and TSTP (TRUSIGHT™ Tumor Panel).

[0026] Figure 9 is a graph showing analysis conducted with data from sites that tested two lots of the control at least once or one lot at least twice. Detection is indicated in dark squares and absence light squares.

[0027] Figure 10 is a graph showing the mean number and mean percentage SNPs detected for CHPv2 and TTP.

[0028] Figure 11 is a graph showing the mean number and mean percentage of SNPs detected for CHPv2 and TACP.

[0029] Figure 12 shows a read length histogram following sequencing. [0030] Figure 13 provides data comparing the number of sequence reads vs the position of the read in a given sequence.

[0031] Figure 14 shows the results of qPCR assays with amplicons of varying lengths targeting the MegaMix 2 plasmid. If fragment length is greater than amplicon length, it will be detected by qPCR.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Provided herein are compositions, methods, kits, plasmids, and cells comprising nucleic acid reference sequences and variants of a reference sequence. The compositions disclosed herein have a variety of uses, including but not limited to, assay optimization, validation, and calibration; peer-to- peer comparison; training and PT/EQA, QC monitoring, reagent QC, and system installation assessment.

[0033] There is a recognized need in the market for flexible, reliable control materials for NGS testing (see Assuring the Next Quality of Next-Generation Sequencing in Clinical Laboratory Practice; Next Generation Sequencing: Standardization of Clinical Testing (Nex-SToCT) Working group Principles and Guidelines, Nature Biotechnology, doi:10.1038/nbt.2403; and ACMG Clinical laboratory standards for next generation sequencing, American College of Medical Genetics and Genomics, doi: 10.1038/gim.2013.92). This disclosure provides such control materials.

[0034] This disclosure relates to control reagents representing reference sequences and / or variants thereof (e.g., mutations) that may be used for various purposes such as, for instance, assay validation / quality control in sequencing reactions (e.g., next generation sequencing (NGS) assays).

Traditional metrics used to characterize the quality of a sequencing reaction include, for instance, read length, minimum quality scores, percent target-mapped reads, percent pathogen-specific reads, percent unique reads, coverage levels, uniformity, percent of non-covered targeted bases and / or real-time error rate. Parameters that may affect quality include, for instance, the types and / or number of analytes being monitored (e.g., the types and number of polymorphisms (single or multiple nucleotide polymorphisms (SNPs, MNPs)), insertions and / or deletions, amplicons, assay contexts and / or limits of detection), sample type (e.g., mammalian cells, infectious organism, sample source), commutability (e.g., validation across multiple technology platforms and / or types of screening panels being utilized), sample preparation (e.g., library preparation type / quality and / or type of sequencing reaction (e.g., run conditions, sequence context)), and / or other parameters. Those of ordinary skill in the art realize, for instance, that the quality of such reactions may vary between laboratories due to subtle differences in guidelines, the metrics and parameters mentioned above, the reference standards used, and the fact that many NGS technologies are highly complex and evolving. This disclosure provides quality control reagents that may be used in different laboratories, under different conditions, with different types of samples, and / or across various technology platforms to confirm that that assays are being carried out correctly and that results from different laboratories may be reliably compared to one another (e.g,. that each is of suitable quality). In some embodiments, the problem of confirming the quality of a sequencing reaction is solved using a multiplex control comprising multiple nucleic acid fragments, each representing a different variant of a reference sequence.

[0035] In certain embodiments, a control reagent for use in sequencing reactions is provided. The control reagent may comprise one or more components that may be used alone or combined to assess the quality of a particular reaction. For instance, some assays are carried out to identify genetic variants present within a biological sample. The control reagents described herein may also provide users with the ability to compare results between laboratories, across technology platforms, and / or with different sample types. For instance, in some embodiments, the control reagent may represent a large number of low percentage (e.g., low frequency) variants of different cancer-related genes that could be used to detect many low percentage variants in a single assay and / or confirm the reliability of an assay. The control reagent could be used to generate numerous data points to compare reactions (e.g., run-to-run comparisons). The control reagent may be used to determine the reproducibility of variant detection over time across multiple variables. The control reagent may be used to assess the quality of a sequencing run (i.e., that the instrument has sufficient sensitivity to detect the included variants at the given frequencies). The control reagent may also be used to differentiate between a proficient and a non-proficient user by comparing their sequencing runs, and /or to differentiate the quality of reagents between different lots. The control reagent may also aid in assay validation studies, as many variants are combined in one sample material. This obviates the need for multiple samples containing one or two variants each, and greatly shortens the work and time required to validate the assay.

[0036] The control reagent typically comprises one or more nucleic acid (e.g., DNA, RNA, circular RNA, hairpin DNA and/or RNA) fragments containing a defined reference sequence of a reference genome (defined as chromosome and nucleotide range) and / or one or more variants of the reference sequence. The source material for the variants may be genomic DNA, synthetic DNA, and combinations thereof. A variant typically includes nucleotide sequence variations relative to the reference sequence. The variant and reference sequence typically share at least 50% or about 75- 100% (e.g., any of about 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99%) sequence identity. In some embodiments, however, the identity shared may be significantly less where, for example, the variant represents a deletion or insertion mutation (either of which may be up to several kilobases or more). An exemplary deletion may be, for instance, recurrent 3.8 kb deletion involving exons 17a and 17b within the CFTR gene as described by Tang, et al. (J. Cystic Fibrosis, 12(3): 290-294 (2013) (describing a c.2988 + 1616_c.3367 +

356del3796ins62 change, flanked by a pair of perfectly inverted repeats of 32 nucleotides)). In some embodiments, variants may include at least one of a single nucleotide polymorphism (SNP), one or more multiple nucleotide polymorphism(s) (MNV), insertion(s), deletion(s), copy number variation(s), gene fusion(s), duplication(s), inversion(s), repeat polymorphism(s), homopolymer(s), non-human sequence(s), or any combination thereof. Such variants (which may include by reference any combinations) may be included in a control reagent as part of the same or different components. The reference sequence(s) and / or variants may be arranged within a control reagent as cassettes.

[0037] Cassettes contains a reference sequence or variant adjoined and / or operably linked to one or more restriction enzyme site(s), sequencing primer(s) site, and / or η3ΐ ίη-&πηη¾ site(s). In some embodiments, it may be useful to include different types of sequences adjacent to each cassette; for instance, it may be useful to design one cassette to be adjacent to a restriction enzyme and / or a hairpin sequence. Doing so may help prevent problems such as cross-amplification between adjacent fragments/cassettes. As such, each reference sequence and / or variant may be releasable and / or detectable separate from any other reference sequence and / or variant. The typical cassette may be about 400 bp in length but may vary between 50-20,000 bp (e.g., such as about any of 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 800, 900, 1000, 2500, 5000, 7500, 10000, 12500, 15000, 17500, or 20000 bp). Each control reagent may comprise one or more cassettes, each representing one or more reference sequence(s) and / or variant(s) (e.g., each being referred to as a "control sequence"). Each reference sequence and / or variant may be present in a control sequence and / or control reagent at percentage of about any of 0.1% to 100% (e.g., about any 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%). For instance, a control sequence or control reagent that is 100% reference sequence or variant would be a reagent representing only one reference sequence or variant. Similarly, a control sequence or control reagent comprising 50% of a variant would be a control reagent representing only up to two reference sequences and / or variants. The remaining percentage could consist of other sequences such as control sequences and the like.

[0038] In certain embodiments a T7 or other promoter can be present upstream of each cassette. This allows for massively parallel transcription of many gene regions. This technique facilitates construction of a control containing equivalent amounts of each target sequence. When there are equivalent amounts of many targets, ease of use of the control is increased. For example, contamination in a control in a patient sample would be easier to detect because all transcripts would show up in the contaminated sample. It is highly unlikely for patient samples to contain, e.g., a large number of fusion transcripts, such an assay result would signal the user that that a

contamination issue is present. This is in contrast to a situation in which only one transcript is present at a much higher abundance in a contaminated sample—which could lead to the contaminant being mistaken for a true positive signal. The ability to construct a control with equivalent amounts of each target sequence eliminates the potential for this type of error.

[0039] In certain embodiments, the reference sequence(s) and / or variants may be adjoined and / or operably linked to one or more different restriction enzyme sites, sequencing primer site(s), and / or η3ΐ ίη-&πηη¾ sites. As described above, certain designs may be used to prevent problems such as cross-amplification between reference sequences and / or variants. In some embodiments, the control sequences and / or cassettes may optionally be arranged such that the same are releasable from the control reagent. This may be accomplished by, for instance, including restriction enzyme (RE) sites at either end of the control sequence. A control reagent may therefore be arranged as follows: RE site/control sequence/RE site. The RE sites may be the same and / or different from one another. The RE sites in one cassette may also be the same and / or different to those present in any other cassette. As such, the control sequences may be released from the cassette as desired by the user by treating the control sequence with one or more particular restriction enzymes.

[0040] In some embodiments, the control reagent may comprise multiple components that may be used together. In certain embodiments, the multiple components comprise a first and a second component which may be plasmids comprising different control sequences and / or different arrangements of the same control sequences. Thus, the components may represent the same or different reference sequences and / or variants. Such components may be used together as a panel, for instance, such that a variety of reference sequences and / or variants may be assayed together. Where the reference sequences and / or variants are the same, each component may include those variants in different cassette arrangements and / or forms. In some embodiments, the multiple components may comprise a first component representing one or more SNP variants and a second component representing one or more multiple nucleotide polymorphism(s), insertion(s), deletion(s), copy number variation(s), gene fusion(s), duplication(s), inversion(s), repeat polymorphism(s), homopolymer(s), and / or non-human sequence(s). The components may be the same or different types of nucleic acids such as plasmids, with each comprising the same or different variants of one or more reference sequences arranged as described herein or as may be otherwise determined to be appropriate by one of ordinary skill in the art. In some embodiments, different types of plasmids may be combined to provide a multi-component control reagent representing many different reference sequences and / or variants.

[0041] Plasmids can be quantified by any known means. In one embodiment, quantitation of each plasmid is performed using a non-human 'xeno' digital PCR target sequence. The exact copy number of the plasmid is determined. The exact copy number of genomic DNA is also determined (obtained by quantification of genomic target site(s)). With this information, controls can be accuratley and reproducibly developed that contain all targets/variants within a tight frequency range.

[0042] The variants may be contained within the control reagent as DNA fragments, each containing a defined sequence derived from a reference genome (defined as chromosome and nucleotide range) with one or more variations (e.g., nucleotide differences) introduced into the fragment. A variant may be, for instance, a sequence having one or more nucleotide sequence differences from the defined sequence (e.g., a reference sequence). For instance, an exemplary reference sequence may comprise "hostpots" suitable for modification. Such hotspots may represent nucleotides and / or positions in a reference sequence that occur in nature (e.g., mutations observed in cancer cells). One or more of such hotspots may be modified by changing one or more nucleotides therein to produce a control sequence (or portion thereof) that may be incorporated into a control reagent. For example, modification of the exemplary epidermal growth factor receptor (EGFR) Ex 19 reference sequence to produce control sequences (Hotspots 1, 2, 3, 4, 5) is shown below (see also, Figure 1):

[0043] Wild Type (e.g., EGFR Exl9) CCAAGCTC (SEQ ID NO: 1)... AGGATCTTGA (SEQ ID NO: 2)... AACTGAATTC (SEQ ID NO: 3)... AAAAAG (SEQ ID NO:

4)... ATC AAAGTGC (SEQ ID NO: 5) (400 bp)

[0044] Hotspot ID 1 CCAATCTC (SEQ ID NO: 6)... AGGATCTTGA (SEQ ID NO:

2)...AACTGAATTC (SEQ ID NO: 3)...AAAAAG (SEQ ID NO: 4)... ATC AAAGTGC (SEQ ID

NO: 5) [0045] Control Sequence Contains Multiple Hotspots CCAATCTC (SEQ ID NO: 6;

HOTSPOT ID 1 ) ... AGG AACTTG A (SEQ ID NO: 7; HOTSPOT ID 2)...AACTCAATTC (SEQ ID NO: 8; HOTSPOT ID 3)...ATAAAG (SEQ ID NO: 9; HOTSPOT ID 4) ... ATGAAAGTGC (SEQ ID NO: 10; HOTSPOT ID 5). This exemplary control sequence thereby represents multiple EGFR variants (e.g., Hotspot IDs 1, 2, 3, 4, 5, etc.) A control reagent may comprise multiple control sequences, each representing one or more variants of the same or different reference sequences. Any number of variants may be represented by a control sequence, and any number of control sequences may be included in a control reagent. A control reagent may comprise, for instance, a number of variants such that the all possible variants of a particular reference sequence are represented by a single control reagent. For instance, the control reagent may comprise mutliple SNPs, MNPs, deletions, insertions and the like, each representing a different variant of the reference sequence. Additional, exemplary, non-limiting variants are shown in Tables 1 A and IB and Table 6.

[0046] Control reagents may also be designed to represent multiple types of control sequences. For instance, control reagents may be designed that represent multiple types of reference sequences and / or variants thereof (which may be found in control sequences alone or in combination). Exemplary categories of control sequences for which the control reagents described herein could have relevance include not only the aforemetioned cancer-related areas but also fields of inherited disease, microbiology (e.g., with respect to antibiotic resistance mutations, immune-escape related mutations), agriculture (e.g,. plant microbe and / or drug resistance-related mutations), livestock (e.g., mutations related to particular livestock traits), food and water testing, and other areas.

Exemplary combinations (e.g., panels) of cancer-related reference sequences that may be represented by a particular control reagent (or combinations thereof) are shown in Table 2.

[0047] The control reagents and methods for using the same described herein may provide consistent control materials for training, proficiency testing and quality control monitoring. For instance, the control reagents may be used to confirm that an assay is functioning properly by including a specific number of representative sequences and / or variants thereof that should be detected in an assay and then calculating the number that were actually detected. This is exemplified by the data presented in Table 3:

[0048] As illustrated in Table 3, a "bad run" is identified where the number of variants detected does not match the number of variants expected to be detected (e.g., included in the assay). As shown in the exemplary assay of Table 3, if a particular control reagent (or combination thereof) used in an assay includes 15 representative sequences and / or variants thereof, all 15 should be detected if the assay is properly carried out. If less than 15 of these control sequences are not detected, the assay is identified as inaccurate (e.g., a "Bad Run"). If all 15 of the sequences are detected, the assay is identified as accurate (e.g., a "Good Run"). Variations of this concept are also contemplated herein, as would be understood by those of ordinary skill in the art.

[0049] In certain embodiments, the control reagent may be prepared by mixing variant DNA fragments (e.g., as may be incorporated into a plasmid) with genomic DNA or synthesized DNA comprising "wild-type" (e.g., non-variant) sequence. Such sequence may be obtained from or present in control cells (e.g., naturally occurring or engineered / cultured cell lines). In some embodiments, the wild-type sequence may be included on a DNA fragment along with the variant sequence, or the variant sequences may be transfected into and / or mixed with cells (e.g., control cells). In certain embodiments, such mixtures may be used to prepare formalin-fixed, paraffin- embedded (FFPE) samples (e.g., control FFPE samples), for example. For instance, in some embodiments, the control reagent may be prepared and tested by designing a control sequence (e.g., an amplicon) comprising a representative sequence and / or variant thereof; designing restriction sites to surround each amplicon; synthesizing a nucleic acid molecule comprising a cassette comprising the amplicon and the restriction sites; and, incorporating the cassette into a plasmid backbone. The construct may then be tested by sequencing it alone (e.g, providing an expected frequency of 100%) or after mixing the same with, for example, genomic DNA at particular expected frequencies (e.g., 50%). Such constructs may also be mixed with cells for various uses, including as FFPE controls.

[0050] In certain embodiments, the control reagents described herein can also be used to provide a frequency ladder. A frequency ladder is composed of many variants at different frequencies. In some embodiments, the control reagent could be used to provide an "ladder" in, for example, 5% increments of abundance (e.g., about any of 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% abundance). For example, the ladder could be constructed by taking a single sample with many different variants present at high (e.g., 80% allele frequency) and making dilutions down to low frequencies. Alternatively, the ladder could be a single sample containing variants at different frequencies. The ladder could be used as a reference for many sample types, including somatic variants at low abundance (e.g., tumor single nucleotide polymorphisms), or germline variants present at, as a non-limiting example, about 50% abundance. Such a ladder may also be used to determine instrument limits of detection for many different variants at the same time. This saves users time in finding materials containing one to a few variants and resources for testing because all variants are present in a single sample rather than many. An example is provided in Table 8:

[0051] As shown in Table 8, a ladder was constructed by diluting a sample containing 555 variants starting at approximately 50% frequency down to ~3% frequency. The ladder was tested in duplicate using the Ion AMPLISEQ® Cancer Hotspot Panel v2 using the Ion Torrent PERSONAL GENOME MACHINE® (PGM). The frequencies for 35 of the variants are reported for each sample tested. The shaded cells indicate that the variant was not detected. Such data could be used to establish the limit of detection for each variant.

[0052] The ladder could be used across many platforms, including Sanger sequencing and next generation platforms, and both RUO and IVD applications could benefit from use of this standard. The frequency ladder could also serve as internal controls in sequencing reactions, much like the lkb DNA ladder serves as a reference in almost every agarose gel. As an example, one design would provide five unique and five identical sequences as shown in Figures 2 and 3. As shown therein, in one sequence position, there is a variant present in only one of these ten sequences. At a second position, the variant is present in two of the ten sequences. At the third position, the variant is present in three of the sequences, and so on. This would yield variants at 10% frequency increments from 0-100%. This is a simplified example and random intervening sequences may be necessary to prevent sequencing artifacts. The product could take any suitable form such as an oligonucleotide (e.g., PCR fragment or synthetic oligonucleotide), plasmids, or one plasmid with concatenated sequences separated by identical restriction enzyme sites (Fig. 3). An advantage of having all sequence variants on one plasmid is that the relative levels of all ten sequences within a mixture would be well controlled; during manufacturing, the plasmid could be cleaved between each sequence with the same enzyme, giving rise to ten fragments at equal ratios. As would be understood by those of ordinary skill in the art, derivations of such a ladder could include different variant types (e.g., insertions and / or deletions), every nucleotide change could be incorporated into the design (e.g., A->C, A->T, etc.), and / or smaller increments could provide fine-tuned

measurement at abundances lower than 10%. For instance, a second plasmid with other mutations could be added to the initial plasmid at a one to nine ratio, yielding variants at even lower frequencies. Such a low frequency sequencing ladder could be an essential control when measuring somatic mutations that appear, for example, at <10% abundance. In some embodiments, such a sequencing ladder may comprise multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage (e.g., about any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10% or more). Each species may comprise, for instance, any suitable number of nucleotides (e.g., about any of 5, 10, 20, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, or 500). Each species may also comprise a

homopolymer sequence of at least 3 nucleotides. In some embodiments, the nucleic acid of the species is DNA, and these may be encoded on vectors such as plasmids and / or by and / or within cells. In some embodiments, each species may comprise a nucleic acid bar code that may be unique to each species. Methods comprising sequencing the nucleic acid species to calibrate a sequencing instrument, to obtain data for sequencing instrument development work, including algorithm development, base calling, variant calling, and / or verify that an instrument is functioning properly (e.g., IQ/OQ/PQ) are also contemplated.

[0053] One of ordinary skill in the art would understand that the control reagents described herein are broadly useful in a variety of sequencing systems and / or platforms. For instance, the control reagents described herein may be used in any type of sequencing procedure including but not limited to Ion Torrent semiconductor sequencing, Ulumina MISEQ®, capillary electrophoresis,

microsphere-based systems (e.g., Luminex), Roche 454 system, DNA replication-based systems (e.g., SMRT by Pacific Biosciences), nanoball- and / or probe-anchor ligation-based systems (Complete Genomics), nanopore-based systems and / or any other suitable system.

[0054] One of ordinary skill in the art would also understand that the control reagents described herein are broadly useful in a variety of nucleic acid amplification-based systems and / or platforms. The control reagents described herein may used in and / or with any in vitro system for multiplying the copies of a target sequence of nucleic acid, as may be ascertained by one of ordinary skill in the art. Such systems may include, for instance, linear, logarithmic, and/or any other amplification method including both polymerase-mediated amplification reactions (such as polymerase chain reaction (PCR), helicase-dependent amplification (HDA), recombinase-polymerase amplification (RPA), and rolling chain amplification (RCA)), as well as ligase-mediated amplification reactions (such as ligase detection reaction (LDR), ligase chain reaction (LCR), and gap-versions of each), and combinations of nucleic acid amplification reactions such as LDR and PCR (see, for example, U.S. Patent 6,797,470). Such systems and / or platforms may therefore include, for instance, PCR (U.S. Patent Nos. 4,683,202; 4,683,195; 4,965,188; and/or 5,035,996), isothermal procedures (using one or more RNA polymerases (see, e.g., PCT Publication No. WO 2006/081222)), strand displacement (see, e.g., U.S. Patent No. RE39007E), partial destruction of primer molecules (see, e.g., PCT Publication No. WO 2006/087574)), ligase chain reaction (LCR) (see, e.g., Wu, et al., Genomics 4: 560-569 (1990)), and/or Barany, et al. Proc. Natl. Acad. Sci. USA 88:189-193 (1991)), Q RNA replicase systems (see, e.g., PCT Publication No. WO 1994/016108), RNA transcription-based systems (e.g., TAS, 3SR), rolling circle amplification (RCA) (see, e.g., U.S. Patent No. 5,854,033; U.S. Patent Application Publication No. 2004/265897; Lizardi et al. Nat. Genet. 19: 225-232 (1998); and or Baner et al. Nucleic Acid Res., 26: 5073-5078 (1998)), and / or strand displacement amplification (SDA) (Little, et al. Clin. Chem. 45:777-784 (1999)), among others. These systems, along with the many other systems available to the skilled artisan, may be suitable for use with the control reagents described herein.

[0055] In one embodiment, a control reagent may be designed and tested using one or more of the steps below:

• designing a control sequence (e.g., an amplicon) comprising a representative sequence of a particular gene of interest and / or variants thereof (e.g., those targeted by commercially- available NGS tests such as the AMPLISEQ Cancer Hotspot Panel v2, and / or the TRUSEQ Amplicon Cancer Panel);

• identifying sequence from a genome reference source (e.g., Genome Reference Consortium Human Reference 37 (GRCh37)) encompassing the amplicon;

• designing a cassette comprising an ~400 bp sequence comprising the amplicon surrounded by (e.g., 5' and 3') the genomic sequence identified in step b);

• designing restriction sites to surround each cassette prepared in step c) (e.g., where one

version may additionally include sequences that create a hairpin when the DNA is single- stranded);

• synthesizing a nucleic acid molecule comprising the cassette of step c) and restriction sites of step d) using a common vector (e.g., pUC57) (e.g., "plasmid VI");

• preparing a second plasmid (e.g., "plasmid V2") comprising multiple fragments of the gene of interest (and / or variants thereof) with a hairpin structure and a restriction site between each region;

• optionally, linearizing the variant sequences contained within plasmids VI and / or V2 with a restriction enzyme;

• mixing the variants with genomic DNA (e.g., wild-type gDNA) at a particular expected

variant frequency (e.g., approximately 50%);

• optionally, testing the "variant sequence" alone (e.g., providing an expected variant

frequency of 100%); • performing variant detection using NGS.

[0056] In certain embodiment, individual cassettes can be synthesized for all genes of interest and combined with wild type. In certain embodiments, a cassette can be designed with a plurality of variants, which do not interfere with the detection of variants near or adjacent thereto.

[0057] In some embodiments, NGS may be performed using the Ion Personal Genome Machine (PGM) by first constructing libraries following the user manuals for the Ion AMPLISEQ® Library Preparation Manual with AMPLISEQ® Cancer Hotspot Panel v2 reagents; preparing template- positive Ion sphere particles (ISPs) and enriching the same using the Ion OneTouch2 instrument following the Ion PGM Template OT2 200 Kit Manual; sequencing using the Ion PGM Sequencing 200 Kit v2 Manual or Sequencing on the Illumina MISEQ® following the TRUSEQ® Amplicon Cancer Panel user manual or the Illumina MiSeq® user manual; and, performing data analysis for PGM using the Torrent Variant Caller v3.4 and v3.6, and for MISEQ® using the MISEQ® Reporter v2.3).

[0058] The reagents and methods described herein may be used in a variety of settings with a variety of samples. For instance, these reagents and methods may be used to analyze biological samples such as serum, whole blood, saliva, tissue, urine, dried blood on filter paper (e.g, for newborn screening), nasal samples, stool samples or the like obtained from a patient and / or preparations thereof (e.g., FFPE preparations). In some embodiments, control preparations comprising the control reagents described herein may be provided.

[0059] This disclosure further relates to kits comprising one or more control reagents described herein. The kits may be used to carry out the methods described herein or others available to those of orindary skill in the art along with, optionally, instructions for use. A kit may include, for instance, control sequence(s) including multiple reference sequences and / or variations thereof in the form of, for instance, one or more plasmids. In some embodiments, the kit may contain a combination of control sequences organized to provide controls for many variations of one or more reference sequences. In some embodiments, the variations may relate to an oncogene that is diagnostic for a particular cancer. In some embodiments, for instance, the kit may comprise control reagents and / or control samples (e.g., tissue samples) known to cover the breadth of mutations known for a particular cancer. In some embodiments, the variations of the marker are variations of a mutation in a gene that are prognostic for the usefulness of treating with a drug. In some

embodiments, the marker or markers are for a particular disease and / or a variety of diseases (e.g., cancer, infectious disease). In some embodiments, the control reagent(s) may be included in a test to ascertain the efficacy of a drug in testing for the presence of a disease and / or progression thereof. In some embodiments, the kit may comprise control reagents for testing for a series of diseases that have common characteristics and/or symptoms (e.g., related diseases). In some embodiments, the marker may have unknown significance but may otherwise be of interest to the user (e.g., for basic research purposes). The kit may also include a container (e.g., vial, test tube, flask, bottle, syringe or other packaging system (e.g., include injection or blow-molded plastic containers) into which one or more control reagents may be placed / contained, and in some embodiments, aliquoted). Where more than one component is included in the kit, it will generally include at least one second, third or other additional container into which the additional components can be separately placed. Various combinations of components may also be packaged in a single container. The kits may also include reagent containers in close confinement for commercial sale. When the components of the kit are provided in one and / or more liquid solutions, the liquid solution comprises an aqueous solution that may be a sterile aqueous solution. As mentioned above, the kit may also include instructions for employing the kit components as well as the use of any other reagent not included in the kit.

Instructions may include variations that may optionally be implemented. The instructions may be provided as a separate part of the kit (e.g., a paper or plastic insert or attachment) or as an internet- based application. In some embodiments, the kit may control reagents relating to between any number of reference sequences and / or variants thereof which may be detected alone or in combination with one another (e.g., a multiplex assay). In some embodiments, the kit may also comprise at least one other sample containing a defined amount of control reagent and "control" test cell admixed such that the same may provide a reference point for the user. Kits may further comprise one or more of a polymerase and/or one or more oligonucleotide primers. Other variations and arrangements for the kits of this disclosure are contemplated as would be understood by those of ordinary skill in the art.

[0060] Thus, in some embodiments, the disclosure provides a nucleic acid molecule or mixture of nucleic acid molecules comprising multiple variants of a reference sequence, each variant sequence may optionally be releasable from the nucleic acid molecule. In certain embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises variants releasable from the nucleic acid molecule using a restriction enzyme.

[0061] In some embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises at least one single nucleotide polymorphism (SNP), multiple nucleotide polymorphisms (MNP), insertion, deletion, copy number variation, gene fusion, duplication, inversion, repeat polymorphism, homopolymer of a reference sequence, and / or a non-human sequence. In some embodiments, the nucleic acid molecule or mixture of nucleic acid molecules comprises at least 5 variants. In certain embodiments, at least 15, 20, 30, 50, 100, 200, 300 400, 700, 1000 variants are present. In yet other embodiments, greater than 1000 variants are present. In some embodiments, each variant is present (e.g., in the sample being tested) at a high or low-frequency. For instance, in certain embodiments, each variant may be present at a frequency of 1%, 5%, 10%, 15%, 20%, 30%, 40% or 50% or more. In other embodiments, each variant may be present at a frequency of less than 50%, less than 40%, less than 20%, less than 15%, less than 10%, less than 5%, less than 3%, less than 1%, less than 0.5%, less than 0.1%, and any integer in between.

[0062] An advantage of the disclosed control materials is that the "truth" of a sample is known. There are currently no reference materials for which absolute frequency (i.e, the truth) is known, that is, the actual frequency of a given variant or combination of variants present are not known. In contrast, in the disclosed control materials, the actual frequency of variants is known.

[0063] Attendant to the teachings of this disclosure, standardized control materials for next generation sequencing (NGS) assays can be produced. Issues such as variant call differences between sites, variability of reagents across instruments, variation introduced by diverse

bioinformatics pipelines and filters, run-to-run and lab-to-lab variability can be identified and resolved and/or obviated utilizing the control materials.

[0064] A further advantage is that the control materials disclosed herein can comprise any number and type of variants, including insertions and deletions of differing lengths, large numbers of SNPs, etc. No other control material exists that provide such diversity.

[0065] The variants can be any of interest. There is no limit provided herein with respect to the type and number of variants that can be utilized in the current disclosure.

[0066] In certain embodiments, modified nucleotides can be utilized as variants. In certain embodiments, methylation can be detected. For example, CpG methylation can be utilized as a biomarker variant.

[0067] This disclosure also provides reagents and methods for confirming the validity of a sequencing reaction by including a known number of representative sequences and / or variants thereof in a mixture comprising a test sample potentially comprising a test nucleic acid sequence and sequencing the nucleic acids in the mixture, wherein detection of all of the representative sequences and / or variants in the mixture indicates the sequencing reaction was accurate. The representative sequences and / or variants may be of the type described herein. Compositions comprising the same are also provided. The pre-determined percentage may be, for instance, about 1, 5 or 10%. And each species may be from, for instance, 20-500 nucleotides. Each species may comprise a homopolymer sequence of at least 3 nucleotides. The nucleic acids may be DNA. Each species may possess a nucleic acid barcode that may be unique to each species. The nucleic acid species described herein may be used to calibrate a sequencing instrument, for instance. Kits comprising such species, optionally further comprising one or more polymerases and / or one or more oligonucleotide primers are also provided. Plasmids and / or cells comprising multiple nucleic acid species wherein the nucleic acid sequence of each species differs from its neighbor species by a predetermined percentage are also provided.

[0068] It is to be understood that the descriptions of this disclosure are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. Also, the use of "comprise", "contain", and "include", or modifications of those root words, for example but not limited to, "comprises", "contained", and "including", are not intended to be limiting. Use of "or" means "and/or" unless stated otherwise. The term "and/or" means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, "X and/or Y" can mean "X" or "Y" or "X and Y". Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and any value or value range therebetween unless otherwise specifically stated. For example, "from 0.2 to 0.5" may mean 0.2, 0.3, 0.4, and 0.5; ranges therebetween such as 0.2-0.3, 0.3 - 0.4, 0.2 - 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 - 0.39; and the like. The term "about" or "approximately" may refer the ordinary meaning of the term but may also indicate a value or values within about any of 1-10 percent of the listed value.

[0069] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirely for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term's definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. Certain embodiments are further described in the following examples. These embodiments are provided as examples only and are not intended to limit the scope of the claims in any way.

[0070] Aspects of this disclosure may be further understood in light of the following examples, which should not be construed as limiting the scope of the disclosure in any way.

EXAMPLES

Example 1

[0071] An exemplary control reagent was prepared and tested as described below:

a) amplicons were designed comprising the fragments shown in Tables 1-3;

b) genomic sequences were selected to encompass each amplicon (the selected genomic sequences being the chromosome and nucleotide positions of the reference genome corresponding to the 5' nucleotide of the forward and reverse primers for each amplicon and all the sequence between these two nucleotides);

c) a cassette was designed comprising an ~400 bp EGFR sequence comprising the amplicon surrounded by (e.g., 5' and 3') the genomic sequence identified in step b) (the reference sequence is added in roughly equally amounts to each end of the region defined in step b) to comprise a ~400 bp region);

d) restriction enzyme and other sites were designed to each cassette prepared in step c) (e.g., where one version may additionally include sequences that create a hairpin when the DNA is single- stranded; the restriction enzymes being chosen such that the sequences of interest are not digested but simply released from the control reagent) as shown below:

EGFR VI**

EGFR_1-Clal- EGFR_2-HindIII-EGFR_3-SmaI-EGFR_4-XhoI-EGFR_5-NotI-EGFR_6/7- EGFR_8 **EGFR_1, etc. represt EGFR variants; restriction enzyme sites for Clal, Hindlll, Smal, Xhol and Not I enzymes were positioned between variants.

EGFR V2***

EGFR_4-HP(7)- Clal -EGFR_5-HP(7)-HindIII-EGFR_6/7-HP(9)-Smal -EGFR 8

***Hairpin 7 (HP(7)): GGGGGGGTTTTCCCCCCC (SEQ ID NO: 11); HindIII=HindIII RE site;

Hairpin 9 (HP(9)): GGGGGGGGGAACCCCCCCCC (SEQ ID NO: 12); SmaI=SmaI RE site e) the cassette of step d) was incorporated into a common vector (pUC57) (e.g., plasmid VI) by automated synthesis of oligonucleotides on solid-phase synthesizers followed by ligation of overlapping oligonucleotides;

f) a second plasmid (e.g., "plasmid V2") comprising multiple fragments of the gene of interest (and / or variants thereof) with a hairpin structure and a restriction site between each region (e.g., as in exemplary construct EGFR V2 above and Table 4) was also prepared by automated synthesis of oligonucleotides on solid-phase synthesizers followed by ligation of overlapping oligonucleotides ;

g) the variant sequences (Tables 4-6) contained within plasmids VI and / or V2 were then linearized Hindlll;

h) the variants were then mixed with genomic DNA (e.g., wild-type gDNA) at a particular expected variant frequency (e.g., approximately 50%) (plasmid DNA and human embryonic kidney (HEK-293) genomic DNA were quantified using a fluorometer (QUBIT®) to determine the concentration; plasmid and genomic DNA were then mixed together to obtain a 1 : 1 molecular ratio (50% variant frequency));

i) the "variant sequences" were then tested alone to provide an expected variant frequency of 100%) to confirm sequencing; and,

j) variants of step h) were detected by NGS using the Ion Personal Genome Machine (PGM) and Ulumina MiSeq (results are presented in Table 7).

Example 2

FFPE-Embedded Controls

[0072] The results of monitoring assays using FFPE-embedded controls are presented in Figs. 4-7. As shown therein, FFPE-embedded control reagents may be used to monitor variant detection, including low frequency variants (e.g., RBI as indicated by "C" in the figures). Variants may be tracked by the amplicon per se, GC content, sequence context, and / or variant type as desired by those of ordinary skill in the art.

[0073] Each embodiment disclosed herein may be used or otherwise combined with any of the other embodiments disclosed. Any element of any embodiment may be used in any embodiment.

Although the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention. In addition, modification may be made without departing from the essential teachings of the invention.

Example 3

555-variant control performance across multiple test sites

[0074] A control sample was constructed that contained 555 variants from 53 different genes and tested with the Ion AMPLISEQ® Cancer Hotspot Panel v2 (CHPv2), TRUSEQ® Amplicon Cancer Panel (TSACP) and the TRUSIGHT® Tumor Panel. For each panel, two lots of the AcroMetrix® Oncology Hotspot Control were tested in duplicate, in at least two sites. Additional sites only tested one of the lots at least twice or both lots once. Sources of variation between sites may include different instruments, operators and general workflows. Also, variation in bioinformatics pipelines may have contributed significantly to variation in performance results.

[0075] Figure 8 shows performance across different sites and panels. The average number of variants of different types detected in the ACROMETRIX® Oncology Hotspot Control are reported by site and grouped by panel. Note: The total number of variants of each type is different for each panel. See Figures 8-11.

[0076] To assess the detection of specific variants across different panels, twenty-two clinically- relevant variants that were targeted by three panels were selected. Figure 9 shows detection of 22 selected variants across panels. Analysis was conducted with data from sites that tested two lots of the control at least once or one lot at least twice. Detection is indicated in dark squares and absence indicated in light squares. Site-to-site differences are apparent, even amongst those utilizing the same library preparation method, indicating the likelihood of the bioinformatics pipeline having an impact on performance.

Example 4

[0077] Performance of the control material comprising 555 variants is shown in Table 9, wherein SNV (single nucleotide variant), MNV (multiple nucleotide variant), DEL (deletion), INS

(insertion), for CHPv2 (AMPLISEQ® Cancer Hotspot Panel v2), TSACP (TRUSEQ® Amplicon cancer panel), and TSTP (TRUSIGHT® tumor panel) are shown. A variant was considered to be covered by the test method if the variant was positioned between the upstream and downstream primers. A variant was considered detected if it was detected in at least one run of the control. Sanger sequencing was performed on the synthetic DNA prior to dilution with genomic DNA. Variants detected in the genomic DNA were confirmed using publicly available whole genome sequencing information for GM24385.

Example 5

[0078] The control materials provided herein can be used for rapid cell line generation by transiently transfecting plasmids and/or RNA into cells and incorporating such cells into a formalin-fixed paraffin-embedded (FFPE) block for use as a control. Methods for generating FFPE control are provided in US Patent Application Publication No. 2014/0335533 which is incorporated herein by reference in its entirety for all purposes. Accordingly, FFPE material was generated by directly introducing nucleic acids into cells after cell growth and processed into FFPE material. This reduces the time to generate a mutant cell material from 7 months to 1 day, representing significant time and cost savings. Also, by introducing nucleic acid after cell growth, many toxic combinations that can inhibit cell growth or lead to cell death can be avoided. This also simplifies the process of growing and storing cells as one cell line can accommodate hundreds of mutations versus the 10+ engineered cell lines that would be required for the same number of mutations. The reagents and methods provided herein allow for the generation of, for example, a single cell containing one or more predetermined nucleic acid sequences containing one or more predetermined mutations. The reagents and methods provided herein permit the generation of any cell line containing an unlimited number of plasmids or RNA transcripts. Further, the reagents and methods provided herein do not require the integration of non-native nucleic acids into the genome of an engineered cell line.

[0079] This method has been demonstrated to be feasible by transfecting either DNA or RNA into human embryonic kidney (HEK 293) cells. For the DNA study, non-growing HEK 293 cells were transfected with eight (8) different DNA fragments simultaneously, each about 6-14 kb long and containing approximately 50 different mutations each. Lipofectamine 2000 was used for transfection. The cells were subsequently mixed with a polymer and processed into FFPE material. DNA from the FFPE material was extracted and was tested using the Ion Torrent AmpliSeq Cancer Hotspot Panel v2. Over 300 hotspot variants were detected from sequencing. Table 10 and Table 11 provide data showing the results of the DNA transfection method. It is understood that methods provided herein can be used with any technique suitable for transferring nucleic acids in to a cell. In general, a transfection reagent is a compound or compounds that bind(s) to or complex(es) with oligonucleotides and polynucleotides, and mediates their entry into cells. The transfection reagent also mediates the binding and internalization of oligonucleotides and polynucleotides into cells. Examples of transfection reagents include cationic liposomes and lipids, polyamines, calcium phosphate precipitates, histone proteins, polyethylenimine, and polylysine complexes. It has been shown that cationic proteins like histones and protamines, or synthetic polymers like polylysine, polyarginine, polyornithine, DEAE dextran, polybrene, and polyethylenimine may be effective intracellular delivery agents, while small polycations like spermine are ineffective. Typically, the transfection reagent has a net positive charge that binds to the oligonucleotide's or polynucleotide's negative charge. The transfection reagent mediates binding of oligonucleotides and polynucleotides to cells or via ligands that bind to receptors in the cell. For example, cationic liposomes or polylysine complexes have net positive charges that enable them to bind to DNA or RNA. Polyethylenimine, which facilitates gene transfer without additional treatments, probably disrupts endosomal function itself. Other vehicles are also used, in the prior art, to transfer genes into cells. These include complexing the nucleic acids on particles that are then accelerated into the cell. This is termed "biolistic" or "gun" techniques. Other methods include electroporation, microinjection, liposome fusion, protoplast fusion, viral infection, and iontophoresis.

[0080] In addition, to assess whether the Fast FFPE method produced fragmented DNA as expected for a typical FFPE material, qPCR assays that amplify different lengths of DNA were used to compare the FFPE DNA to intact plasmid DNA. This study demonstrated that the FFPE DNA was more fragmented than the plasmids.

[0081] For the RNA study, two different EML4-ALK in-vitro fusion gene RNA transcripts were generated and transfected into non-growing HEK 293 using Lipofectamine 2000. The cells were subsequently processed into FFPE material. RNA from the FFPE material was extracted and tested using two qPCR assays that specifically amplify the EML4-ALK fusion. The FFPE material was positive for both transcripts. Table 12 provides data indicating that RNA transcripts of EML4-ALK fusions are detectable following transfection.

[0082] These reagents and methods provided herein demonstrate that FFPE material containing hundreds of different DNA or RNA mutations can be created by a single transfection and that the nucleic acid extracted from such materials shows aspects of true FFPE material.

[0083] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

TABLE 1A

Exemplary Hotspot (HS) Variants

Gene name Mutation ID Mutation CDS Mutation Description Chr Start End

MPL 27286 C.1514G>A Substitution - Missense 1 43814979 43814979

MPL 18918 C.1544G>T Substitution - Missense 1 43815009 43815009

MPL 27290 C.1555G>A Substitution - Missense 1 43815020 43815020

NRAS 584 C.182A>G Substitution - Missense 1 115256529 115256529

NRAS 1332933 C.174A>G Substitution - coding silent 1 115256537 115256537

NRAS 577 c.52G>A Substitution - Missense 1 115258730 115258730

NRAS 564 c.35G>A Substitution - Missense 1 115258747 115258747

NRAS 24850 c.29G>A Substitution - Missense 1 115258753 115258753

ALK 28056 c.3824G>A Substitution - Missense 2 29432664 29432664

ALK 28055 C.35220A Substitution - Missense 2 29443695 29443695

MSH6 13399 c.3246G>T Substitution - coding silent 2 48030632 48030632

MSH6 13395 c.3261delC Deletion - Frameshift 2 48030647 48030647

MSH6 1021299 c.3300G>A Substitution - coding silent 2 48030686 48030686

IDH1 28746 c.395G>A Substitution - Missense 2 209113112 209113112

IDH1 1404902 c.388A>G Substitution - Missense 2 209113119 209113119

IDH1 96922 c.367G>A Substitution - Missense 2 209113140 209113140

ERBB4 48362 c.2791G>T Substitution - Missense 2 212288954 212288955

ERBB4 169572 c.2782G>T Substitution - Nonsense 2 212288963 212288964

ERBB4 232263 C.1835G>A Substitution - Missense 2 212530083 212530084

ERBB4 573362 C.18280A Substitution - Missense 2 212530090 212530091

ERBB4 1405173 C.1784A>G Substitution - Missense 2 212530134 212530135

ERBB4 1614287 C.1089T>C Substitution - coding silent 2 212576809 212576810

ERBB4 110095 C.1022OT Substitution - Missense 2 212576876 212576877

ERBB4 573356 C.1003G>T Substitution - Missense 2 212576895 212576896 ERBB4 1405181 c.909T>C Substitution - coding silent 2 212578347 212578348

ERBB4 160825 c.885T>G Substitution - Missense 2 212578371 212578372

ERBB4 1015994 C.8290A Substitution - Missense 2 212587171 212587172

ERBB4 1251447 C.804OA Substitution - Nonsense 2 212587196 212587197

ERBB4 1405184 c.730A>G Substitution - Missense 2 212589811 212589812

ERBB4 573353 C.704OT Substitution - Missense 2 212589837 212589838

ERBB4 1015997 c.633G>A Substitution - coding silent 2 212589908 212589909

ERBB4 48369 c.542A>G Substitution - Missense 2 212652763 212652764

ERBB4 442267 C.5150G Substitution - Missense 2 212652790 212652791

VHL 14305 c.266T>A Substitution - Missense 3 10183797 10183797

VHL 18080 c.277G>C Substitution - Missense 3 10183808 10183808

VHL 17658 C.2860T Substitution - Nonsense 3 10183817 10183817

VHL 17886 c.296delC Deletion - Frameshift 3 10183827 10183827

VHL 17752 C.3430A Substitution - Missense 3 10188200 10188200

VHL 14312 c.353T>C Substitution - Missense 3 10188210 10188210

VHL 14407 c.388G>C Substitution - Missense 3 10188245 10188245

VHL 14412 c.431delG Deletion - Frameshift 3 10188288 10188288

VHL 17657 C.4720G Substitution - Missense 3 10191479 10191479

VHL 17612 C.4810T Substitution - Nonsense 3 10191488 10191488

VHL 14311 C.4990T Substitution - Missense 3 10191506 10191506

VHL 17837 c.506T>C Substitution - Missense 3 10191513 10191513

MLH1 26085 C.1151T>A Substitution - Missense 3 37067240 37067240

CTNNB1 5677 C.980G Substitution - Missense 3 41266101 41266101

CTNNB1 5662 c.llOOT Substitution - Missense 3 41266113 41266113

CTNNB1 5664 C.121A>G Substitution - Missense 3 41266124 41266124

CTNNB1 5667 C.1340T Substitution - Missense 3 41266137 41266137

F0XL2 33661 C.402OG Substitution - Missense 3 138665163 138665163

PIK3CA 27495 c.35G>A Substitution - Missense 3 178916648 178916648

PIK3CA 27376 c.93A>G Substitution - Missense 3 178916706 178916706

PIK3CA 1420738 C.180A>G Substitution - coding silent 3 178916793 178916793

PIK3CA 1041454 C.210OT Substitution - coding silent 3 178916823 178916823 PIK3CA 27497 c.323G>A Substitution - Missense 3 178916936 178916936

PIK3CA 13570 c.331A>G Substitution - Missense 3 178916944 178916944

PIK3CA 125368 c.344G>T Substitution - Missense 3 178916957 178916957

PIK3CA 1420774 c.536A>G Substitution - Missense 3 178917661 178917661

PIK3CA 21462 C.9710T Substitution - Missense 3 178921489 178921489

PIK3CA 353193 C.1002OT Substitution - coding silent 3 178921520 178921520

PIK3CA 754 C.1035T>A Substitution - Missense 3 178921553 178921553

PIK3CA 1420804 C.1213T>C Substitution - Missense 3 178927450 178927450

PIK3CA 757 C.1258T>C Substitution - Missense 3 178927980 178927980

PIK3CA 1420828 C.1370A>G Substitution - Missense 3 178928092 178928092

PIK3CA 759 C.16160G Substitution - Missense 3 178936074 178936074

PIK3CA 760 C.1624G>A Substitution - Missense 3 178936082 178936082

PIK3CA 763 C.1633G>A Substitution - Missense 3 178936091 178936091

PIK3CA 1420865 C.1640A>G Substitution - Missense 3 178936098 178936098

PIK3CA 778 c.2102A>C Substitution - Missense 3 178938860 178938860

PIK3CA 769 c.2702G>T Substitution - Missense 3 178947827 178947827

PIK3CA 770 c.2725T>C Substitution - Missense 3 178947850 178947850

PIK3CA 328026 c.3110A>G Substitution - Missense 3 178952055 178952055

PIK3CA 775 c.3140A>G Substitution - Missense 3 178952085 178952085

PIK3CA 12464 c.3204_3205insA Insertion - Frameshift 3 178952149 178952150

FGFR3 715 C.7460G Substitution - Missense 4 1803568 1803568

FGFR3 29446 C.7530T Substitution - coding silent 4 1803575 1803575

FGFR3 723 c.850delC Deletion - Frameshift 4 1803672 1803672

FGFR3 716 c.ll08G>T Substitution - Missense 4 1806089 1806089

FGFR3 24842 C.1138G>A Substitution - Missense 4 1806119 1806119

FGFR3 724 C.1150T>C Substitution - Missense 4 1806131 1806131

FGFR3 721 C.11720A Substitution - Missense 4 1806153 1806153

FGFR3 1428724 C.1928A>G Substitution - Missense 4 1807869 1807869

FGFR3 719 C.1948A>G Substitution - Missense 4 1807889 1807889

FGFR3 24802 c.2089G>T Substitution - Missense 4 1808331 1808331

PDGFRA 12418 c.l698_1712dell Complex - deletion inframe 4 55141052 55141066 KDR 1430203 c.3433G>A Substitution - Missense 4 55955112 55955112

KDR 48464 c.2917G>T Substitution - Missense 4 55961023 55961023

KDR 1430212 c.2619A>G Substitution - coding silent 4 55962505 55962505

KDR 32339 c.824G>T Substitution - Missense 4 55979623 55979623

FBXW7 1427592 c.2079A>G Substitution - coding silent 4 153244078 153244078

FBXW7 27083 C.2065OT Substitution - Missense 4 153244092 153244092

FBXW7 732399 C.2033OG Substitution - Nonsense 4 153244124 153244124

FBXW7 34018 c.2001delG Deletion - Frameshift 4 153244156 153244156

FBXW7 27913 C.1580A>G Substitution - Missense 4 153247222 153247222

FBXW7 30599 C.1576T>C Substitution - Missense 4 153247226 153247226

FBXW7 30598 C.1558G>A Substitution - Missense 4 153247244 153247244

FBXW7 34016 C.1451G>T Substitution - Missense 4 153247351 153247351

FBXW7 22974 C.1436G>A Substitution - Missense 4 153247366 153247366

FBXW7 22965 C.1394G>A Substitution - Missense 4 153249384 153249384

FBXW7 22986 C.1338G>A Substitution - Nonsense 4 153249440 153249440

FBXW7 161024 C.1322G>T Substitution - Missense 4 153249456 153249456

FBXW7 22973 C.11770T Substitution - Nonsense 4 153250883 153250883

FBXW7 22971 C.8320T Substitution - Nonsense 4 153258983 153258983

FBXW7 1052125 c.744G>T Substitution - Missense 4 153259071 153259071

APC 18979 c.2543_2544insA Insertion - Frameshift 5 112173834 112173835

APC 18852 C.26260T Substitution - Nonsense 5 112173917 112173917

APC 19230 c.2639T>C Substitution - Missense 5 112173930 112173930

APC 19330 C.26560T Substitution - Nonsense 5 112173947 112173947

APC 19065 c.2752G>T Substitution - Nonsense 5 112174043 112174043

APC 13872 C.32860T Substitution - Nonsense 5 112174577 112174577

APC 1432250 c.3305A>G Substitution - Missense 5 112174596 112174596

APC 1432260 c.3435A>G Substitution - coding silent 5 112174726 112174726

APC 41617 c.3700delA Deletion - Frameshift 5 112174991 112174991

APC 1432280 c.3795A>G Substitution - coding silent 5 112175086 112175086

APC 19072 C.38710T Substitution - Nonsense 5 112175162 112175162

APC 18960 C.3880OT Substitution - Nonsense 5 112175171 112175171 MET 695 c.3785A>G Substitution - Missense 7 116423456 116423456

MET 691 c.3803T>C Substitution - Missense 7 116423474 116423474

SMO 13145 C.5950T Substitution - Missense 7 128845101 128845101

SMO 13147 c.970G>A Substitution - Missense 7 128846040 128846040

SMO 216037 C.12340T Substitution - Missense 7 128846398 128846398

SMO 13146 C.1604G>T Substitution - Missense 7 128850341 128850341

SMO 13150 C.1918A>G Substitution - Missense 7 128851593 128851593

BRAF 476 C.1799T>A Substitution - Missense 7 140453136 140453136

BRAF 471 C.1790T>G Substitution - Missense 7 140453145 140453145

BRAF 467 C.1781A>G Substitution - Missense 7 140453154 140453154

BRAF 462 C.1742A>G Substitution - Missense 7 140453193 140453193

BRAF 450 C.1391G>T Substitution - Missense 7 140481417 140481417

BRAF 27986 C.1380A>G Substitution - coding silent 7 140481428 140481428

BRAF 1448625 C.1359T>C Substitution - coding silent 7 140481449 140481449

BRAF 6262 C.1330OT Substitution - Missense 7 140481478 140481478

EZH2 37028 C.1937A>T Substitution - Missense 7 148508727 148508727

FGFR1 1292693 C.8160T Substitution - coding silent 8 38282147 38282147

FGFR1 187237 C.4480T Substitution - Missense 8 38285864 38285864

FGFR1 1456955 c.421A>G Substitution - Missense 8 38285891 38285891

FGFR1 601 C.3740T Substitution - Missense 8 38285938 38285938

JAK2 12600 C.1849G>T Substitution - Missense 9 5073770 5073770

JAK2 27063 C.1860OA Substitution - Missense 9 5073781 5073781

CDKN2A 12479 c.358G>T Substitution - Nonsense 9 21971000 21971000

CDKN2A 12476 C.3410T Substitution - Missense 9 21971017 21971017

CDKN2A 12547 c.330G>A Substitution - Nonsense 9 21971028 21971028

CDKN2A 13489 c.322G>T Substitution - Missense 9 21971036 21971036

CDKN2A 12504 C.2470T Substitution - Missense 9 21971111 21971111

CDKN2A 12475 C.2380T Substitution - Nonsense 9 21971120 21971120

CDKN2A 13281 c.205G>T Substitution - Nonsense 9 21971153 21971153

CDKN2A 12473 C.1720T Substitution - Nonsense 9 21971186 21971186

GNAQ 1110323 C.1002OT Substitution - coding silent 9 80336317 80336317 ATM 21826 c.2572T>C Substitution - Missense 11 108138003 108138003

ATM 22507 c.3925G>A Substitution - Missense 11 108155132 108155132

ATM 21920 C.5044OT Substitution - Missense 11 108170479 108170479

ATM 218294 C.51520G Substitution - Missense 11 108170587 108170587

ATM 49005 C.5178-1G>T Unknown 11 108172374 108172374

ATM 172204 C.51880T Substitution - Nonsense 11 108172385 108172385

ATM 21918 c.5224G>C Substitution - Missense 11 108172421 108172421

ATM 12792 C.5380OT Substitution - coding silent 11 108173640 108173640

ATM 1183962 c.5476T>G Substitution - Missense 11 108173736 108173736

ATM 21922 c.5821G>C Substitution - Missense 11 108180945 108180945

ATM 12951 c.7325A>C Substitution - Missense 11 108200958 108200958

ATM 12791 c.7996A>G Substitution - Missense 11 108204681 108204681

ATM 21636 c.8084G>C Substitution - Missense 11 108205769 108205769

ATM 1235404 C.8095OA Substitution - Missense 11 108205780 108205780

ATM 22481 c.8174A>T Substitution - Missense 11 108206594 108206594

ATM 1183939 c.8624A>G Substitution - Missense 11 108218045 108218045

ATM 22485 C.8668OG Substitution - Missense 11 108218089 108218089

ATM 21930 c.8839A>T Substitution - Missense 11 108225590 108225590

ATM 21626 c.9023G>A Substitution - Missense 11 108236087 108236087

ATM 1351060 c.9054A>G Substitution - coding silent 11 108236118 108236118

ATM 21624 C.91390T Substitution - Nonsense 11 108236203 108236203

KRAS 41307 c.491G>A Substitution - Missense 12 25362805 25362805

KRAS 19940 c.351A>C Substitution - Missense 12 25378647 25378647

KRAS 554 C.183A>C Substitution - Missense 12 25380275 25380275

KRAS 546 C.175G>A Substitution - Missense 12 25380283 25380283

KRAS 1169214 C.IOIOT Substitution - Missense 12 25398207 25398207

KRAS 14208 C.104OT Substitution - Missense 12 25398215 25398215

KRAS 521 c.35G>A Substitution - Missense 12 25398284 25398284

KRAS 507 c.24A>G Substitution - coding silent 12 25398295 25398295

PTPN11 13011 C.181G>T Substitution - Missense 12 112888165 112888165

PTPN11 13013 c.205G>A Substitution - Missense 12 112888189 112888189 TP53 11073 C.1024OT Substitution - Nonsense 17 7574003 7574003

TP53 11286 C.1015G>T Substitution - Nonsense 17 7574012 7574012

TP53 11071 C.1009OT Substitution - Missense 17 7574018 7574018

TP53 11514 C.IOOIOT Substitution - Missense 17 7574026 7574026

TP53 11354 C.9910T Substitution - Nonsense 17 7576855 7576855

TP53 44823 c.981T>G Substitution - Nonsense 17 7576865 7576865

TP53 46088 c.963A>G Substitution - coding silent 17 7576883 7576883

TP53 10786 C.9490T Substitution - Nonsense 17 7576897 7576897

TP53 10663 C.9160T Substitution - Nonsense 17 7577022 7577022

TP53 10710 c.892G>T Substitution - Nonsense 17 7577046 7577046

TP53 10863 C.8330T Substitution - Missense 17 7577105 7577105

TP53 10660 c.818G>A Substitution - Missense 17 7577120 7577120

TP53 10662 c.743G>A Substitution - Missense 17 7577538 7577538

TP53 6932 c.733G>A Substitution - Missense 17 7577548 7577548

TP53 10812 C.7220T Substitution - Missense 17 7577559 7577559

TP53 10725 c.701A>G Substitution - Missense 17 7577580 7577580

TP53 10758 c.659A>G Substitution - Missense 17 7578190 7578190

TP53 44317 c.653T>A Substitution - Missense 17 7578196 7578196

TP53 10667 c.646G>A Substitution - Missense 17 7578203 7578203

TP53 43947 c.614A>G Substitution - Missense 17 7578235 7578235

TP53 10738 c.542G>A Substitution - Missense 17 7578388 7578388

TP53 10808 c.488A>G Substitution - Missense 17 7578442 7578442

TP53 10739 c.481G>A Substitution - Missense 17 7578449 7578449

TP53 10670 c.469G>T Substitution - Missense 17 7578461 7578461

TP53 10801 c.404G>A Substitution - Missense 17 7578526 7578526

TP53 11582 c.395A>G Substitution - Missense 17 7578535 7578535

TP53 11462 c.388C>G Substitution - Missense 17 7578542 7578542

TP53 44226 C.380OT Substitution - Missense 17 7578550 7578550

TP53 44985 c.375+17G>A Unknown 17 7579295 7579295

TP53 43904 c.375G>A Substitution - coding silent 17 7579312 7579312

TP53 10716 c.329G>T Substitution - Missense 17 7579358 7579358

SMARCB1 51386 c.566_567insl9 Insertion - Frameshift 22 24145547 24145548

SMARCB1 993 C.601OT Substitution - Nonsense 22 24145582 24145582

SMARCB1 999 C.607OA Substitution - Missense 22 24145588 24145588

SMARCB1 1057 c.ll48delC Deletion - Frameshift 22 24176357 24176357

TABLE IB Exemplary Copy Number Variants (CNV)

Gene Name Chromosome Start End

ERBB2 chrl7 37845134 37845207

ERBB2 chrl7 37852282 37852381

ERBB2 chrl7 37860184 37860303

ERBB2 chrl7 37871503 37871582

ERBB2 chrl7 37876682 37876784

ERBB2 chrl7 37884464 37884584

ERBB2 chrl7 37854903 37855025

ERBB2 chrl7 37884065 37884183

ERBB2 chrl7 37866483 37866606

ERBB2 chrl7 37880963 37881086

KRAS chrl2 25378600 25378682

PDGFRA chr4 55140973 55141093

TABLE 2

Control Reagent*

Sequence A B C D E F G H

1 CSF1R APC APC APC APC CSF1R APC APC

2 EGFR EGFR CSF1R CSF1R CSF1R EGFR EGFR CSF1R

3 FBXW7 FBXW7 EGFR FGFR3 EGFR FGFR1 FGFR3 EGFR 4 FGFR3 FGFR3 ERBB4 FLT3 FBXW7 FGFR3 FLT3 FGFR3

5 FLT3 FLT3 FGFR3 KDR FGFR3 FLT3 HRAS FLT3

6 GNA11 KDR FLT3 KRAS FLT3 HRAS IDH1 HRAS

7 HNF1A KRAS HRAS PDGFRA HRAS KDR KDR IDH1

8 HRAS PDGFRA KDR RET KRAS KIT KRAS KRAS

9 PDGFRA PH 3CA KIT STK11 PDGFRA KRAS PDGFRA PDGFRA

10 PIK3CA RET KRAS TP53 RET MET RET PIK3CA

11 RET TP53 PDGFRA - SMAD4 NOTCH1 TP53 RET

12 STK11 - RET - TP53 PDGFRA - STK11

13 TP53 - SMAD4 - - PIK3CA - TP53

14

VHL - TP53 - - SMARCBl - -

15 - - - - - SMO - -

16 - - - - - TP53 - -

[0084] * APC (Adenomatous polyposis coli, deleted in polyposis 2.5 (DP2.5); Chr. 5: 112.04-112.18 Mb; Ref. Seq. NM 000038 and NP 000029), CSF1R (Colony stimulating factor 1 receptor, macrophage colony- stimulating factor receptor (M-CSFR), CD115; Chr. 5, 149.43-149.49 Mb; Ref. Seq. NM 005211 and NM 005202), EGFR (epidermal growth factor receptor; Chr. 7: 55.09-55.32 Mb; RefSeq Nos. NM 005228 and NP 0052219), FBXW7 (F-box/WD repeat-containing protein 7; Chr. 4: 153.24-153.46 Mb; RefSeq. Nos. NM 001013415 andNP_001013433), FGFR1 (Fibroblast growth factor receptor 1, basic fibroblast growth factor receptor 1, fms-related tyrosine kinase-2 / Pfeiffer syndrome, CD331; Chr. 8: 38.27-38.33 Mb; RefSeq. Nos. NM 001174063 and NP OOl 167534), FGFR3 (Fibroblast growth factor receptor 3, CD333; chr. 4: 1.8- 1.81 Mb; RefSeq Nos. NM 000142 andNP_000133), FLT3 (Fms-like tyrosine kinase 3, CD135, fetal liver kinase-2 (Flk2); Chr. 13: 28.58-28.67 Mb; RefSeq Nos. NM_004119 andNP_004110), GNA11 (Guanine nucleotide-binding protein subunit alpha-11; Chr. 19: 3.09-3.12 Mb; RefSeq Nos. NM 002067 and

NP 002058), HNFIA (hepatocyte nuclear factor 1 homeobox A; Chr. 12: 121.42-121.44 Mb; RefSeq Nos. NM 000545 and NP 000536), HRAS (GTPase HRas, transforming protein p21; Chr. 11: 0.53-0.54 Mb; RefSeq Nos. NM 001130442 and NP OOl 123914), IDH1 (Isocitrate dehydrogenase 1 (NADP+), soluble; Chr. 2: 209.1-209.13 Mb; RefSeq Nos. NM 005896 and NP 005887), KDR (Kinase insert domain receptor, vascular endothelial growth factor receptor 2, CD309; Chr. 4: 55.94-55.99 Mb; RefSeq Nos. NM 002253 and NP 002244), KIT (Mast/stem cell growth factor receptor (SCFR), proto-oncogene c-Kit, tyrosine-protein kinase Kit, CD117; Chr. 4: 55.52-55.61 Mb; RefSeq Nos. NM_000222 and NP_000213), KRAS (GTPase KRas, V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; Chr. 12: 25.36-25.4 Mb; RefSeq Nos.

NM 004985-NP 004976), MET (c-Met, MNNG HOS Transforming gene, hepatocyte growth factor receptor; Chr. 7: 116.31-116.44 Mb; RefSeq Nos. NM_000245 and NP_000236), NOTCH1 (Notch homolog 1, translocation-associated (Drosophila); Chr. 9: 139.39-139.44; RefSeq Nos. NM 017617 and NP 060087), PDGFRA (Alpha-type platelet-derived growth factor receptor; Chr. 4: 55.1-55.16 Mb; RefSeq Nos.

NM_006206 and NP_006197), PIK3CA (pi 10a protein; Chr. 3: 178.87-178.96 Mb; RefSeq Nos.

NM 006218 and NP 006209), RET (receptor tyrosine kinase; Chr. 10: 43.57^13.64; RefSeq Nos.

NM_000323 and NP_065681), SMAD4 (Chr. 18: 48.49-48.61 Mb; RefSeq Nos. NM_005359 and

NP 005350), SMARCB1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1; Chr. 22: 24.13-24.18 Mb; RefSeq Nos. NM_001007468 and NP_001007469), SMO (Smoothened; Chr. 7: 128.83-128.85 Mb; RefSeq Nos. NM_005631 and NP_005622), STK11

(Serine/threonine kinase 11, liver kinase Bl (LKBl), renal carcinoma antigen NY-REN- 19; Chr. 19: 1.19- 1.23 Mb; RefSeq Nos. NM 000455 and NP 000446), TP53 (protein 53, tumor protein 53; Chr. 17: 7.57-7.59 Mb; RefSeq Nos. NM 000546 and NP 000537), VHL (Von Hippel-Lindau tumor suppressor; Chr. 3: 10.18- 10.19 Mb; RefSeq Nos. NM_000551 and NP_000542).

[0085] One or more variants of each of these reference sequences may also be represented in each control sequence and / or control reagent. In some embodiments, for instance, multiple variants may be included for each reference sequence. Panels of reference sequences may also be designed to represent particular metabolic, genetic information processing, environmental information processing, cellular process, organismal system, disease, drug development, or other pathways (e.g., KEGG pathways (http://www.genome.jp/kegg/pathway.html, Nov. 8, 2013)). Control reagents such as these may be assayed separately or combined into a single assay. The control reagents may also be designed to include various amounts of each reference sequences and / or variants thereof.

Table 3

Number of Variants Number of Variants

Run ID Detection Rate

Detected Expected

Bad Run 8 15 53%

Bad Run 12 15 80%

Good Run 15 15 100%

Good Run 15 15 100%

Good Run 15 15 100%

Good Run 15 15 100% Number of Variants Number of Variants

Run ID Detection Rate

Detected Expected

Good Run 15 15 100%

Good Run 15 15 100%

Table 4

Table 5

Included variants for each EGFR fragment

EGFR l EGFR 2 EGFR 3 EGFR 4 EGFR 5 EGFR_6_7 EGFR 8

COSM21683 COSM21686 COSM21689 COSM41905 COSM13180 COSM28603 COSM6224

COSM21687 COSM21690 COSM28508 COSM53194 COSM26445 COSM12675

COSM28511 COSM18419 COSM6241 COSM6213

COSM12988 COSM13182 COSM12376 COSM14070

COSM13427 COSM17570 COSM12381 COSM28607

COSM41603 COSM6223 COSM13007 COSM33725

COSM28601 COSM21984 COSM6240 COSM13008

COSM6252 COSM13192 COSM26438

COSM6239 COSM28610

COSM12373 COSM41663

COSM22992

COSM28510

COSM13979 Table 6

Mutation Detail

Mutation Gene Mutation Mutation CDS Mutation AA Mutation Chr Start End GRCh3' ID name ID Description strand

21683 EGFR 21683 c.323G>A p.R108K Substitution 7 55211080 55211080 +

- Missense

21686 EGFR 21686 c.865G>A p.A289T Substitution 7 55221821 55221821 +

- Missense

21687 EGFR 21687 0.866OT p.A289V Substitution 7 55221822 55221822 +

- Missense

21689 EGFR 21689 0.1787OT p.P596L Substitution 7 55233037 55233037 +

- Missense

21690 EGFR 21690 c.l793G>T p.G598V Substitution 7 55233043 55233043 +

- Missense

41905 EGFR 41905 c.2092G>A p.A698T Substitution 7 55241644 55241644 +

- Missense

28508 EGFR 28508 c.2104G>T p.A702S Substitution 7 55241656 55241656 +

- Missense

28511 EGFR 28511 c.2108T>C p.L703P Substitution 7 55241660 55241660 +

- Missense

12988 EGFR 12988 c.2125G>A p.E709K Substitution 7 55241677 55241677 +

- Missense

13427 EGFR 13427 c.2126A>C p.E709A Substitution 7 55241678 55241678 +

- Missense

41603 EGFR 41603 c.2134T>C p.F712L Substitution 7 55241686 55241686 +

- Missense

28601 EGFR 28601 c.2135T>C p.F712S Substitution 7 55241687 55241687 +

- Missense

6252 EGFR 6252 c.2155G>A p.G719S Substitution 7 55241707 55241707 +

- Missense

6239 EGFR 6239 c.2156G>C p.G719A Substitution 7 55241708 55241708 +

- Missense

12373 EGFR 12373 0.2159OT p.S720F Substitution 7 55241711 55241711 +

- Missense

22992 EGFR 22992 c.2161G>A p.G721S Substitution 7 55241713 55241713 +

- Missense

28510 EGFR 28510 c.2162G>C p.G721A Substitution 7 55241714 55241714 +

- Missense

13979 EGFR 13979 c.2170G>A p.G724S Substitution 7 55241722 55241722 +

- Missense

13180 EGFR 13180 0.2188OT p.L730F Substitution 7 55242418 55242418 +

- Missense

53194 EGFR 53194 0.2197OT p.P733S Substitution 7 55242427 55242427 +

- Missense Mutation Gene Mutation Mutation CDS Mutation AA Mutation Chr Start End GRCh3' ID name ID Description strand

18419 EGFR 18419 c.2200G>A p.E734K Substitution 7 55242430 55242430 +

- Missense

13182 EGFR 13182 c.2203G>A p.G735S Substitution 7 55242433 55242433 +

- Missense

17570 EGFR 17570 0.2222OT p.P741L Substitution 7 55242452 55242452 +

- Missense

6223 EGFR 6223 c.2235_2249dell5 p.E746_A750deELREA Deletion - 7 55242465 55242479 +

In frame

21984 EGFR 21984 c.2281G>T p.D761Y Substitution 7 55242511 55242511 +

- Missense

28603 EGFR 28603 c.2293G>A p.V765M Substitution 7 55248995 55248995 +

- Missense

26445 EGFR 26445 C.2300OT p.A767V Substitution 7 55249002 55249002 +

- Missense

6241 EGFR 6241 c.2303G>T p.S768I Substitution 7 55249005 55249005 +

- Missense

12376 EGFR 12376 c.2307_2308insGCCAGCGTG p.V769_D770insASV Insertion - 7 55249009 55249010 +

In frame

12381 EGFR 12381 c.2319_2320insAACCCCCAC p.H773_V774insNPH Insertion - 7 55249021 55249022 +

In frame

13007 EGFR 13007 c.2335_2336GG>TT p.G779F Substitution 7 55249037 55249038 +

- Missense

6240 EGFR 6240 0.2369OT p.T790M Substitution 7 55249071 55249071 +

- Missense

13192 EGFR 13192 c.2428G>A p.G810S Substitution 7 55249130 55249130 +

- Missense

28610 EGFR 28610 c.2441T>C p.L814P Substitution 7 55249143 55249143 +

- Missense

41663 EGFR 41663 c.2462T>C p.I821T Substitution 7 55249164 55249164 +

- Missense

6224 EGFR 6224 c.2573T>G p.L858R Substitution 7 55259515 55259515 +

- Missense

12675 EGFR 12675 c.2575G>A p.A859T Substitution 7 55259517 55259517 +

- Missense

6213 EGFR 6213 c.2582T>A p.L861Q Substitution 7 55259524 55259524 +

- Missense

14070 EGFR 14070 c.2588G>A p.G863D Substitution 7 55259530 55259530 +

- Missense

28607 EGFR 28607 c.2603A>G p.E868G Substitution 7 55259545 55259545 +

- Missense

33725 EGFR 33725 c.2609A>G p.H870R Substitution 7 55259551 55259551 +

- Missense

13008 EGFR 13008 0.2612OG p.A871G Substitution 7 55259554 55259554 +

- Missense Mutation Gene Mutation Mutation CDS Mutation AA Mutation Chr Start End GRa^

ID name ID Description strand

26438 EGFR 26438 c.2620G>A p.G874S Substitution 7 55259562 55259562 +

- Missense

Table 7

Results of EGFR plasmid

EGFR Plasmid VI EGFR Plasmid V2

Mutation Ion AmpliSeq Illumina Ion AmpliSeq Illumina

ID CHP2 TruSeq CHP2 TruSeq

21683 Called Called Not Included Not Included

21686 Called Called Not Included Not Included

21687 Called Called Not Included Not Included

21689 Called Not Targeted Not Included Not Included

21690 Called Called Not Included Not Included

41905 Called Not Called** Called Not Called**

28508 Called Not Called** Called Not Called**

28511 Called Not Called** Called Not Called**

12988 Not Called Not Called** Not Called Not Called**

13427 Called Not Called** Called Not Called**

41603 Called Not Called** Called Not Called**

28601 Called Not Called** Called Not Called**

6252 Called Not Called** Called Not Called**

6239 Called Not Called** Called Not Called**

12373 Not Called Not Called** Not Called Not Called**

22992 Called Not Called** Called Not Called**

28510 Called Not Called** Called Not Called**

13979 Called Not Called** Called Not Called**

13180 Called Called Called Called

53194 Called Called Called Called

18419 Called Called Called Called

13182 Called Called Called Called

17570 Called Called Called Called

6223 Not Called* Called Not Called* Called EGFR Plasmid Vl EGFR Plasmid V2

Mutation Ion AmpliSeq Illumina Ion AmpliSeq Illumina ID CHP2 TruSeq CHP2 TruSeq

21984 Called Called Called Called

28603 Called Called Called Called

26445 Called Not Called Called Not Called

6241 Called Not Called Called Not Called

12376 Called Not Called Called Not Called

12381 Called Not Called Called Not Called

13007 Called Called Called Called

6240 Called Called Called Called

13192 Called Called Called Called

28610 Called Called Called Called

41663 Called Not Called Called Not Called

6224 Called Not Called Called Not Called

12675 Called Not Called Called Not Called

6213 Called Not Called Called Not Called

14070 Called Not Called Called Not Called

28607 Called Not Called Called Not Called

33725 Called Not Called Called Not Called

13008 Called Not Called Called Not Called

26438 Called Not Called Called Not Called

*Mutation not called by software, but manual inspection revealed that the sequence corresponded to the correct mutation

** Variant introduced in primer region of test method

Called: sequence variant noted by analysis software

Not Targeted: sequence variant not included in sequence analyzed by the test method Table 8

63

98

o

O

o

o

o

O

Table 10

Mega Mix Transfection MegaMix Control

Sample Variants Hotspot Variants Sample Variants Hotspot Variants

Meg3 ixTransfection_l_l 423 306 AS SJinal 412 29?

;Vega ixTransfection _„ l_2 426 309 ASMS_Fmal 415 300

Mega ixTransfection_2_l 423 305 ASMS Lot! 407 292

MegaMixTransfection_2_2 423 305 _k ASMS_Lotl 403 291 jV1ega ixTransfection_2_3 425 305

MegaMixTransfection_3_l 422 304

Vlega ixTransfection_3_2 424 306

Vlega ixTransfection_3_3 424 305

Table 1 1

Table 12