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• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
  • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the invention relates to methods for identification of genomic variants.
• the invention further relates to identification of chromosomal anomalies arising from mutation, deletion, substitution, insertion or rearrangement of such gene segments in cancer cells.
• Cancer develops through a series of genetic mutations, with tumor cells acquiring pernicious mutations that eventually lead to metastatic disease.
• the DNA mutations contributing to oncogenesis are not limited to point mutations, but include large chromosomal rearrangements, duplications, and deletions. It has been suggested that recurring mutations are the likely drivers for cancer, and might be viable biomarkers for disease detection and prognosis.
• capturing the DNA breakpoints associated with recurrent deletions is challenging via current targeted sequencing methods due to variability in the deletion size (up to 2 mb) and exact position. The effort is particularly challenging when the exact loci of breakpoints which make up the deletion in a given patient— and therefore the sequence of the resulting variant polynucleotide from the wild-type genomic nucleic acid— is unknown.
• AML acute myeloid leukemia
• Such loss of DNA may also contribute to cancer progression.
• many human cancers frequently delete chromosome 9p21 -22 locus containing ⁇ , CDKN2A, and CDKN2B genes.
• the locus encodes INK4 proteins (p ⁇ 5 1NK4b , p ⁇ 6 INK4a ) that inhibit cyclin-dependent kinases, CDK4 and CDK6, and p ⁇ 4 ARF , which inactivates MDM2 and thereby regulating p53.
• INK4 proteins p ⁇ 5 1NK4b , p ⁇ 6 INK4a
• CDK4 and CDK6 cyclin-dependent kinases
• p ⁇ 4 ARF p ⁇ 4 ARF
• DNA lesions in cancer are the ideal markers for monitoring cancer progression and therapeutic response in patients.
• DNA lesions with high frequency and large deviation from normal DNA serve as the best targets because they are detected more often.
• INK4A and ARF are adjacent tumor suppressor genes that are frequently deleted (-33%) in the early neoplastic stages of different types of cancer.
• DNA lesions can be used to (a) detect/characterize tumor DNA in individuals and (b) monitor tumor burden during or after treatment. It has been demonstrated that identification of BCR-ABL gene fusion at the DNA level in leukemia patients leads to a more sensitive test for measuring tumor burden than current BCR- ABL mRNA tests. Measuring changes in tumor burden during therapeutic treatment is critical for checking therapy effectiveness and deciding to continue treatment. It has been found that circulatory tumor DNA had the highest correlation with tumor burden and greater dynamic range than current standard of care CA 15-3 biomarker and circulatory tumor cell counting in metastatic breast cancer.
• SNP-arrays hybridization microarrays (SNP-arrays), which are still widely used in clinics, are capable of calling copy number variation, from which deletions and gene
• amplifications can be inferred.
• the technology is only reliable with homogeneous samples and only reports low resolution boundaries estimates, insufficient for performing tumor burden monitoring assays.
• a challenge remains how to detect DNA markers, specifically, somatic structural variations, in a complex patient sample containing a mixture of tumor DNA and germline DNA. This is particularly challenging when the exact breakpoints are needed for quantitative DNA assays.
• described herein are methods for more reliable and expansive capture of chromosomal rearrangement events while still using simple molecular biology techniques.
• the present invention is based in part on a method used to detect genetic variations or markers specific to tumor cells.
• the method targets structural variation (SV) breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, this method can validate SVs called by whole exome/genome sequencing and hybridization arrays.
• the invention further provides for the identification patient specific markers, methods to monitor the patients therapeutic response, remission and relapse.
• the invention additionally provides for kits for the detection of such variants.
• Deletions in a cancer genome are all the result of one breakpoint in a normal genome, while inversions, and translocations in a cancer genome are the result of two breakpoints. Therefore, according to the invention, forward oligos are selected from one DNA stretch and reverse oligos are selected from another DNA stretch. If both breakpoints occur in the targeted DNA stretches, then the chromosomal rearrangement will be selectively amplified.
• a computational pipeline was developed for specialized oligo selection to allow for long range PCR amplification and maximize coverage of potential breakpoints.
• Rearranged sequences that are amplified are then sequenced and analyzed.
• Structural variants SVs or breakpoints, like CDKN2A deletion, can be utilized as patient-specific tumor biomarkers.
• AmBre target SV breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, AmBre can validate SVs called by whole exome/genome sequencing and hybridization arrays.
• AmBre involves a PCR-based approach to amplify the DNA segment containing a SVs breakpoint and then confirms breakpoints using DNA sequencing technology.
• primers tiling specified target regions are carefully selected with a simulated annealing algorithm to minimize off-target amplification and maximize efficiency at capturing all possible breakpoints within the target regions.
• PCR amplicons are combined without barcoding and long-read sequenced simultaneously. The algorithm efficiently separates reads based on breakpoints. Each read group supporting the same breakpoint corresponds with an amplicon and a consensus amplicon sequence is called.
• AmBre can target SVs where DNA harboring the breakpoints are present in 1 : 1000 mixtures.
• the invention provides for a high sensitivity method for detecting a variant polynucleotide of unknown nucleotide sequence believed to differ from the wildtype nucleotide sequence of a nucleic acid molecule of interest, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the method comprising: a) computational design of a multiplicity of primers to detect one or more breakpoint loci in the wildtype nucleic acid of interest; b) use of the multiplicity of primers in multiplex PCR to amplify any variant polynucleotides present in the sample; c) analysis of the sequence of each multiplex PCR products; d) detection of the variant polynucleotide in the sample; and e) high through-put sequence analysis of the variant polynucleotide to confirm the differences in nucleotide sequence in the variant as compared to the sequence of the wild-type nucleic acid
• the variant is a mutation, deletion, insertion, substitution or genomic rearrangement.
• the genomic rearrangement is an inversion, deletion, translocation, alteration or a duplication.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately 1-20 kb around the locus of interest and the innermost primers is separated by approximately at least 15 - 100 kb.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 6 kp around the locus of interest and the innermost primers is separated by
• the ratio of primer spacing to locus of interest is 3 kp to 380 kb. In a further aspect, the ratio of primer spacing to locus of interest is 6 kp to 80 kb. In an additional aspect, the ratio of primer spacing to locus of interest is 3 kp to 80 kb. In one aspect, the multiplicity of primers are selected for minimum cost in loss of assay sensitivity, wherein the cost is calculated by the equation:
• the analysis of the multiplex PCR products comprises: a) sequencing the nucleotide of interest and b) analyzing the sequence by: i) sequence alignment to wild- type nucleotide sequence, ii) alignment trimming, iii) clustering of breakpoints, and iv) confirming the variant sequence.
• the multiplicity of primers consists of approximately 2-80 primers. In a preferred aspect, the multiplicity of primers consists of approximately 16 primers.
• the variant is detected in multiple samples simultaneously. In one aspect, the method can detect multiple variants simultaneously.
• the high through-put sequence analysis is single molecule sequence analysis.
• the high through-put sequence analysis has a high error rate.
• the high through-put sequence analysis error rate is comprised of up to about 20% insertion error rates and deletion rates and/or up to about 5% substitution error rates.
• the method comprises a further step of prognosing, determining progression of cancer, predicting a therapeutic regimen or predicting benefit from therapy in a subject having a disease based on the detection of a variant.
• the disease is cancer.
• the cancer is selected from the group consisting of a carcinoma, sarcoma, leukemia, lymphoma, myeloma, and a CNS tumor.
• the cancer is selected from the group consisting of skin cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, thyroid cancer, ovarian cancer, uterine cancer, breast cancer, cervical cancer, kidney cancer, epithelial carcinoma, squamous cell carcinoma, basal cell carcinoma, melanoma, papilloma, and adenomas.
• the invention provides a kit for detecting a variant polynucleotide having a nucleotide sequence differing from the wildtype nucleotide sequence of a nucleic acid molecule, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the kit comprising a) a multiplicity of primers; and b) algorithms to detect the variant.
• the variant is a mutation, deletion, insertion, substitution or genomic rearrangement.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately 1-20 kb around the locus of interest and the innermost primers is separated by approximately at least 15-100 kb. In an additional aspect, the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 5-15 kb around the locus of interest and the innermost primers is separated by approximately at least 20-80 kb.
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 6 kp around the locus of interest and the innermost primers is separated by approximately at least 20 kb. In a further aspect, the ratio of primer spacing to locus of interest is 6 kp to 80 kb.
• the present invention provides a system for analyzing a variant polynucleotide of unknown nucleotide sequence believed to differ from the wildtype nucleotide sequence of a nucleic acid molecule of interest in a subject, comprising: a) a multiplicity of primers computationally designed to detect one or more breakpoints in the wildtype nucleic acid sequence of the subject; b) a computer -executable algorithm for detecting the variant in a sample from a subject having a cancer, the algorithm comprising computational design of a multiplicity of primers; c) use of the multiplicity of primers in multiplex PCR to amplify any variant polynucleotide present in the sample; d) analysis of the sequence of each multiplex PCR products; e) detection of the variant polynucleotide in the sample; and f) long range sequencing of the variant polynucleotide to confirm the differences in nucleotide sequence in the variant as compared to the sequence of
• system further comprising a machine to perform the multiplex PCR. In another aspect, the system further comprising a machine to sequence the multiplex PCR products. In a further aspect, the system further comprising a machine to perform long range sequencing of a detected variant polynucleotide.
• the invention provides a method for confirming variant polynucleotide of unknown nucleotide sequence which differs from the wildtype nucleotide sequence of a nucleic acid molecule of interest, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the method comprising: a) sequencing the nucleotide of interest; and b) analyzing the sequence by i) sequence alignment to wild-type nucleotide sequence, ii) alignment trimming, iii) clustering of breakpoints, and iv) confirming the variant sequence.
• the high through-put sequence analysis is single molecule sequence analysis.
• the high through-put sequence analysis has a high error rate.
• the high through-put sequence analysis error rate is comprised of up to about 20% insertion error rates and deletion rates and/or up to about 5% substitution error rates.
• Figure 1 shows a standard PAMP tiling design for capture of CDKN2A deletions.
• CDKN2A upstream and downstream breakpoint regions are defined on a germline genome, blue and red lines, respectively.
• Tiled forward primers and reverse primers are spaced Ikb apart (width of hashed boxes) (not to scale with reference). Overlap of blue box and red box on tumor DNA represents a forward and reverse primers are less than 2kb apart and will lead to amplification of tumor DNA
• Figure 2 is a flow chart of the AmBre method with primer designing and long fragment sequence analysis.
• Figure 3 depicts the process of designing, analyzing and selecting primers
• AmBre primer design was used to capture CDKN2A deletion upstream and downstream breakpoints in regions chr9 : 21, 730, 000 - 21, 965,000 and chr9 : 21, 975, 000 - 22, 129, 000 (GRCH37 coordinates), respectively.
• Simulated annealing using different convergence rates is used to select good primer designs with lowest sensitivity cost. The convergence rate that finds the lowest sensitivity cost primer design will depend on the input given to AmBre-design.
• Figure 4 shows the PCR products of AMBRE-16 on cell-lines: A549 (lane 2), CEM (lane 3), Detroit562(lane 4), HeLa(lane 5), MCF7 (lane 6), and T98G (lane 7). 4 ⁇ 1 of lkb GeneRuler in lane 1. Lanes are reactions starting with lOng cell-line genomic DNA. HeLa cells (no CDKN2A deletion) and H 2 0 are negative controls. Arrow denotes weak Detroit562 band; another PCR had stronger amplification and was used for subsequent sequencing.
• Figure 5 shows the aggregates of breakpoints from PacBioTM fragments after sweep line clustering. Only breakpoints with L ⁇ 1000 are displayed for inset boxes. The height of each cluster corresponds with number of fragments supporting the breakpoint (depth of breakpoint coverage).
• Figure 6 shows the breakpoint sequences for A549, CEM, Detroit562, MCF7, and T98G with orthogonal validation chromatogram of MCF7 and T98G. AmBre-analyze captures both breakpoints and nontemplated insert sequence.
• Figure 7 shows the subsampling of 9 primers from the complete AMBRE-68 tiling design results in clean amplification of CDKN2A loss DNA fragments in 6 cell lines. From left to right, lanes contain lkb Plus GeneRuler DNA ladder, PCR products from samples A549 (2.2kb), CEM (5.8kb), MCF7 (3.6kb), MOLT4 (6,8kb), T98G (7.5kb), HEK (Okb), and water (Okb). The expected lengths of each amplicon according to AMBRE-68 design are listed in parentheses. HEK cells (no CDKN2A deletion) and H 2 0 are negative controls.
• Figure 8 depicts successful A549 (arrow) and MCF7 (arrow) CDKN2A deletion amplification with heterogeneity ratios 1 : 1, 1 : 10, 1 : 100, 1 : 1000 (lanes 3-6 for A549 and lanes 10-13 for MCF7) and 16 primers starting with 400ng of gDNA.
• Lane 1 contains lkbp Plus Gene Ruler DNA ladder.
• Lanes 2 and 9 are A549 and MCF7 positive control reactions starting with 20ng of homogenous gDNA.
• Lanes 7,14 are negative control reactions with wild-type DNA and lanes 8, 15 are water negative control reactions with corresponding 16 primer mixes.
• Figure 9 shows a) Fragment-segmentation example for local alignments 1, 2,3, and 4 along a PacBio fragment, b) Triangle representation of adjacent alignments 1, 2, and 3 on G x G plane.
• FIG. 10 Characterizing RUNX1-RUNX1T1 balanced translocation in Kasumi-1.
• Lanes 1,2,4,6 and 8 contain lkb Plus GeneRuler DNA ladder, PCR products from Kasumi-1 Der8 with all 28 primers (3:5Kbp), 14 primer FE U RO (3:5Kbp), 14 primer FO U RO
• Lanes 3,5,7 and 9 contain matching water controls, which show no contamination. Lanes 10,12,14, and 16 contain PCR products from Kasumi-1 Der21 with all 29 primers (2:7Kbp), 15 primer FO U RO (2:7Kbp), 15 primer FE U RO (6: lKbp), and 14 FE U RE (8: lKbp). Gel was loaded with 2ul for lanes 2,3,4,5,10,11,12 and 13, and 4ul for remaining volumes. Reactions with shorter amplicons amplified extremely well and lesser volumes were used for visualization on the gel. The expected amplicon lengths according to the Der8 and Der21 design are listed in parentheses.
• Figure 11 shows DNA helix stability around breakpoints. Using code from BreakSeq pipeline, DNA flexibility or the 6 breaks around proposed non-homologous end joining DNA breaks showed no significant deviation.
• the present invention is based in part on a method used to detect genetic variations or markers specific to tumor cells.
• the method targets structural variation (SV) breakpoints occurring in samples composed of heterogeneous tumor and germline DNA. Additionally, this method can validate SVs called by whole exome/genome sequencing and hybridization arrays.
• the invention further provides for the identification patient specific markers, methods to monitor the patients therapeutic response, remission and relapse.
• variant refers to a polynucleotide having a sequence differing from the wildtype nucleotide sequence of a nucleic acid molecule.
• high through-put sequence analysis or “high through-put sequencing” as used throughout this disclosure refer to a method for obtaining digital readouts of sequence from a DNA sample. Typically, a multiplicity of reads is found from a single sample with lengths ranging from 30-20000bp. Reads with lengths greater than 100 bp are considered long reads.
• Single molecule sequencing is an example of high through-put sequencing in which long nucleotide sequences can be sequence in a single pass without shearing of PCR products. High throughput sequencing may be performed using any appropriate sequencing technology. Examples of such technologies include SangerTM sequencing, SBSTM sequencing and Pacific BiosystemTM sequencing.
• primer or “primers” as used throughout this disclosure refer a an oligonucleotide sequence used as a reagent for PCR.
• oligonucleotides are typically 20 -40 nucleotides in length, but may be longer or shorter. Primers may be modified for detection. Such modifications include biotinylation, labeling with fluorescent dyes or other known labeling methods.
• Wild type DNA refers any DNA which does not have a variant polynucleotide. Wild type DNA would include heterogeneous tumor DNA.
• a mutation is a change of the nucleotide sequence of the genome. Mutations result from unrepaired damage to DNA or to RNA genomes, errors in the process of replication, or from the insertion or deletion of segments of DNA by mobile genetic elements. Mutation can result in several different types of change in sequences. Mutations in genes can either have no effect, alter the product of a gene, or prevent the gene from functioning properly or completely. Mutations can also occur in noncoding regions. Examples of mutations include point mutations, substitutions, insertions or deletions.
• Genomic rearrangement maybe large scale nucleic acid mutations.
• genomic rearrangements include inversion in which the nucleotide orientation is reversed, deletion in which nucleotides are deleted, translocation in which nucleotides are moved to a different chromosomal location, alteration or a duplication of nucleotides.
• genomic rearrangements include CDKN2A deletion, TP53 deletion, PIK3A deletion, EGFR deletion, PTEN deletion, KLF5 deletion, BCR-ABL translocation, RUNX1-RUNX1T1 translocation, RARA-PML translocation, MLLT3-MLL translocation, TMPRSS2-ERG translocation and ERB2 inversion.
• the invention provides advantages over existing technology by, for example: a) using long range polymerases, which provides a higher coverage of genetic lesion variability; and,
• AmBre is based on PCR, where a DNA product is amplified only when a tumor specific genetic lesion is present within a patient's DNA sample.
• the invention improves on Primer Approximation Multiplex PCR (PAMP) methodology but differs in several aspects, including utilization of a long range PCR protocol instead of a standard PCR protocol and in not requiring a microarray hybridization step.
• PAMP is a PCR assay, developed to selectively amplify the tumor DNA sequence containing a structural variation (United States Patent Application No. 12/375,912 relating to PAMP is incorporated herein by reference).
• PAMP has several drawbacks.
• all primers In the multiplexed reaction, all primers must be evenly spaced so as to amplify any deletion in the region and primers cannot dimerize.
• 100 applicable primers In a large (e.g. l OOKbp) region, 100 applicable primers must be identified from a large candidate set of over 5000 potential primers. An exhaustive search of all candidate primer combinations is infeasible (5000 candidate primers and 50 to 100
• PAMP is therefore limited to detecting recurrent structural variations where breakpoints appear in short breakpoint regions ( ⁇ 40kb), as a large number of primers in a multiplex reaction inevitably leads to loss of sensitivity with off-target DNA synthesis and increased spurious primer-primer interactions.
• PAMP detects the amplified product and identifies breakpoints via DNA hybridization arrays which had the additional challenge of designing probes that match the primer designs. AmBre resolves these issues with a three-phase approach (Fig. 2).
• the first phase involves a revised computational approach to designing multiplex primers on discontiguous DNA regions ignoring regions known to not contain breakpoints. This requires some changes to the optimization function and results in a more flexible design with better performance on sparse regions.
• the output of this phase is a multiplicity of primers that can be mixed in a single multiplex reaction.
• the amplified products are sequenced using a system from which analysis allows mixing the amplicons prior to sequencing, with computational separation of breakpoints in the third phase.
• a system from which analysis allows mixing the amplicons prior to sequencing with computational separation of breakpoints in the third phase.
• a non-limiting example of such a system is the Pacific Biosciences RS Platform (PacBioTM).
• the third phase involves a customized analysis of sequenced reads to identify DNA breakpoints for each tumor genome.
• the analysis involves clustering of split mapped reads followed by error correction, and sequence reconstruction around the breakpoint regions.
• AmBre can detect targeted structural variations (potential tumor DNA biomarkers) by identifying deletion breakpoints in the cancer cell lines A549, CEM, Detroit562, MCF7, MOLT4, and T98G, including resolution of previously unidentified CDKN2A breakpoints in MCF7 and T98G.
• Ambre can be used to identify translocations and inversion; e.g., as exemplified herein for RUNX1-RUNX1T1 translocation in the cancer cell line Kasumi-1.
• the invention provides for a high sensitivity method for detecting a variant polynucleotide of unknown nucleotide sequence believed to differ from the wildtype nucleotide sequence of a nucleic acid molecule of interest, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the method comprising: a) computational design of a multiplicity of primers to detect one or more breakpoint loci in the wildtype nucleic acid of interest; b) use of the multiplicity of primers in multiplex PCR to amplify any variant polynucleotides present in the sample; c) analysis of the sequence of each multiplex PCR products; d) detection of the variant polynucleotide in the sample; and e) high through-put sequence analysis of the variant polynucleotide to confirm the differences in nucleotide sequence in the variant as compared to the sequence of the wild-type nucleic acid
• the variant is a mutation, deletion, insertion, substitution or genomic rearrangement.
• the genomic rearrangement is an inversion, deletion, translocation, alteration or a duplication.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 4, 5, 6, 7, 8 kp around the locus of interest and the innermost primers is separated by approximately at least 15, 20, 25, 30, 35, 40, 45, 50, 60, 70 or 80 kb.
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 6, kp around the locus of interest and the innermost primers is separated by approximately at least 20 kb.
• the ratio of primer spacing to locus of interest is 3 kp to 380 kb. In a further aspect, the ratio of primer spacing to locus of interest is 6 kp to 80 kb. In an additional aspect, the ratio of primer spacing to locus of interest is 3 [0053] kp to 80 kb. In one aspect, the multiplicity of primers are selected for minimum cost in loss of assay sensitivity, wherein the cost is calculated by the equation:
• the analysis of the multiplex PCR products comprises: a) sequencing the nucleotide of interest and b) analyzing the sequence by: i) sequence alignment to wild- type nucleotide sequence, ii) alignment trimming, iii) clustering of breakpoints, and iv) confirming the variant sequence.
• the multiplicity of primers consists of approximately 2-80 primers.
• the multiplicity of primers consists of approximately 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 40, 50 or 60 primers.
• the multiplicity of primers consists of approximately 16, primers.
• the variant is detected in multiple samples simultaneously. In one aspect, the method can detect multiple variants simultaneously.
• the method can detect multiple variant polynucleotides in multiple samples simultaneously.
• the high through-put sequence analysis is single molecule sequence analysis.
• the high through-put sequence analysis has a high error rate.
• the high through-put sequence analysis error rate is comprised of up to about 20% insertion error rates and deletion rates and/or up to about 5% substitution error rates.
• the high throughput sequencing is performed with gel electrophoresis. The subject method can be used for both the detection of known variants and for the identification of unknown variants.
• the present invention utilizes unique computational analysis for the design of the multiplicity of primers.
• the primer design used in the subject method are critical.
• the primers must be unique, must not self-hybridize or hybridize with other primers and must be evenly spaced over the desired length of DNA and amplify the non-target locus wild type DNA.
• the input to AmBre-design is a collection of genomic intervals for the forward region, denoted by F, a collection of genomic intervals for the reverse region (R), and parameter d.
• the output is a collection of forward primers in F and reverse primers located in R spaced apart by approximately d.
• AmBre-design has the following steps:
• the candidate list of primers and incompatible primers is used to design an optimal set of primers based on considerations outlined below.
• a primer design P as a subset of candidate primers numbered according to the order of genomic start locations / / , I 2 , , ... / « .
• B denote incompatible primers.
• a cost C(P) was associated with each design, and designs with minimum cost are preferred. The formulation of cost accommodates sparser primer designs and targeting discontiguous regions.
• the cost of the design is a sum of incompatibility costs for each pair, and coverage costs.
• the distance between adjacent primers is bounded by (l -p)d ⁇ Ai(P) ⁇ d.
• a design is penalized if the distances violate these constraints.
• the multiplicity of primers is approximately between 2-40. In other aspects the multiplicity of primers maybe 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60 ,65, 70, 75 or 80. In a preferred aspect the multiplicity of primers is 16.
• locus of interest refers to the nucleotide sequence targeted for detection or identification of a variant or breakpoint.
• the locus of interest is 15 kb to 100 kb in length.
• the locus of interest is 15, 16, 17, 18, 19 ,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95,100, 150, 200, 250, 300, 400, or 500 kb.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately 1-20 kb around the locus of interest and the innermost primers is separated by approximately at least 15-100 kb.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 kb around the locus of interest and the innermost primers is separated by approximately at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or 80 kb.
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 6 kb around the locus of interest and the innermost primers is separated by approximately at least 20 kb.
• the ratio of primers spacing to the locus of interest is approximately 2kb:400 kb-10 kb: 100kb. In further aspects, the ratio of primers spacing to the locus of interest is approximately 3kb:380kb, 3kb:80kb or 6kb to 80 kb.
• the primers are selected for a minimum cost in loss of sensitivity, wherein the cost of a primers is calculated by the equation:
• amplification is performed using long range multiplex PCR, reducing the number of primers required in a single reaction.
• Long Range PCR refers to the amplification of DNA lengths that cannot typically be amplified using routine PCR methods or reagents.
• Multiplex PCR is a modification of polymerase chain reaction in order to target variable products or variability in breakpoints. This process amplifies genomic DNA samples using multiple primers and a temperature-mediated DNA polymerase in a thermal cycler. In contrast to PAMP, the subject method can be used for DNA lengths > 40 kb.
• the subject method can be used for DNA lengths of 40 kb, 50 kb, 60 kb, 70, kb, 80 kb, 90 kb, 100, kb, 150 b, 200 kb, 250 kb, 300 kb, 350 kb, 400 kb, 450 kb or 500 kb.
• Sequencing of the amplified PCR products and computational analysis confirms capture of the variation or breakpoint (for example CDKN2A deletions).
• the subject method utilizes sequencing technology for long reads structural variation calling, and throughput.
• An example of technology suitable sequencing platform is the PacBioTM system from Pacific Biosciences of Menlo Park, California, although other platforms might be utilized.
• the amplified PCR products are sequenced and then aligned to a reference genome to identify variants or breakpoints with the sequence around each breakpoint to identify the variant.
• a breakpoint is defined according to the computational algorithm applied in the AmBre-analyze step as a pair of disjoint coordinates a and b on a reference, and a non- template sequence s (of length t) such that the sample sequence brings a and b together, separated only by the insertion of s.
• the objective of AmBre-analyze is to take as input a collection of sample sequences aligned to the reference genome and output a collection of breakpoints along with the sequence around each breakpoint.
• a unique aspect to the subject application is the detection of a variant
• the subject method can detect a variant polynucleotide in a multiplicity of reads with error rates such as up to 20% insertion and up to 5% substitution rates.
• Ambre-analyze works by (a) alignment trimming, (b) breakpoint or variant clustering of fragments, and (c) consensus sequence generation around each breakpoint or variant, as outlined in Figure 2 and described further below. [0072] Alignment Trimming
• a local alignment is defined as a pair of intervals from the fragment and reference that can be aligned with a small number of edits.
• a split mapped fragment F supports a breakpoint (a, b, s) with two local-alignments (denoted as ( F a , G a ), (F b , G b )).
• G a ends at a
• Gb begins at b
• the fragment segment between F a and Fb is exactly the inserted sequence s (Fig. 2).
• a fragment can span multiple breakpoints, sequence errors can result in spurious incorrect alignments, and the alignments output by standard tools like BLASR, will have uncertain boundaries. Specifically, inaccurate boundaries might result in overlapping consecutive segments F a , Fb.
• the fragment constrains the location of the breakpoint (a, b) to lie in a small region between x, y.
• the subject method uses a unique algorithm for the alignment which works by combining the above objectives into a single objective function, and uses a dynamic programming approach to identify the optimal trimming.
• Local alignments encompassed by other alignments are removed (e.g., 4 in Fig. 9). The remaining alignments were sorted by their location on the fragment, so that alignment i starts before alignment j if and only if i ⁇ j.
• Let b s (i) and b e (i) denote the fragment breaks before the beginning and after the end of alignment i.
• Alignments can be represented on a grid with alignments as rows and fragment positions as columns (Fig. 9).
• An alignment is a series of breaks on the fragment (i.e. (1 , b ) to (1 , bs) in Fig. 9). Alignments are chained together to cover a portion of F exactly once.
• To chain adjacent alignments for each alignment j with an alignment i that terminates before j starts, add a jump from (i, b e (i)) to (j,b s (j)) (for instance (1 , b e ⁇ )) to (3, b s ,(3))).
• any alignment chain covers positions exactly once.
• An alignment chain is scored by summing local alignment scores (Aln[z, u, v] for alignment i for fragment coordinates u to v) and penalizing for jumps between alignments (J(u, v) for alignment u to v).
• a high scoring alignment chain corresponds to trimmed alignments that aligns well and covers most of the fragment.
• the score of a chain is computed using dynamic programming. Let S(j, v) denote the score of the best chain ending at (j, v). Then,
• the optimal trimmed alignment chain is quickly found.
• each jump (F' a , G a ), (F' b ) , G b ), represents a breakpoint estimate (a, b, F - F :).
• the jump from 1 to 3 correspond with breakpoint estimate (xi, y 2 , 6).
• Fragment clustering uses the information from the alignment trimming for multiple fragments to further narrow the breakpoint location.
• a true breakpoint (a, b) is represented by a point
• each split-mapped read (x, y, L) is represented by a triangle of possible breakpoints (a, b) that satisfy (a - x) + (y - b) ⁇ L (Fig. 9b).
• Multiple reads supporting the same breakpoint represent multiple triangles whose intersection reduces the uncertainty in breakpoint determination.
• the split-mapped reads will cluster according to overlap, revealing breakpoints for each experiment sample.
• the subject method uses a fast, customized method to recover the aggregated read clusters for each breakpoint. For example, the method took 2.5 min seconds on a single Desktop core to analyze all local alignments from 52,000 reads from a single PacBioTM SMRT cell experiment.
• Predicted amplicon sequences are generated from the breakpoint estimates. In turn, these templates are supplied as reference sequences for further analysis. For example, the PacBioTM SMRT Analysis Resequencing Protocol analysis protocol culls consensus amplicon sequences by correcting the predicted templates.
• Breakpoint estimates from all fragments supporting the same breakpoint are aggregated into groups using a sweep line algorithm.
• the true breakpoint junctions (a,b) in reference G lies between x.... x + L and y-L ....y, respectively, subject to a - x + y -b ⁇ L.
• L a spacing length on F
• each breakpoint estimate x, y, and L with the above constraints defines a triangle which contains the true breakpoint (a, b) (Fig. 9 and Fig. 5).
• a line sweeps the plane and tracks when breakpoint triangles overlap along the sweep line.
• a cluster is a collection of triangles where each triangle overlaps one or more triangles in the cluster.
• the consensus breakpoint (a, b) for the cluster is the mode of (x, y) estimates (see Fig. 5).
• breakpoints associated with inversions like A549, are captured.
• a breakpoint corresponds with ( -x, y) and (-y, x) or (x, -y) and (y, -x).
• predicted amplicon templates for each cluster are created by joining reference sequence G(6500— a, a) and G(b, b + 6500).
• a reference sequence reconstruction can be performed using, for example, automated system such as the PacBioTM SMRT Analysis 1.4 pipeline for resequencing to refine the amplicon template predictions using all fragments generated from the SMRT cell.
• the resequencing protocol involves running BLASR for mapping followed by Quiver for consensus sequence calling. The protocol accurately recovers the sequence around breakpoints; for example, a consensus amplicon sequence starting at aligned 25 - a and ending at b + 25 matched either sequencing from previous studies or independent Sanger sequencing chromatogram (Fig. 6).
• the subject method may be used to identify variants which are unique to individual subjects, and can be applied to detect variants in mixtures at ratios to wild-type of up to 1 : 100, 1 :500, 1 : 1000, 1 :2500, 1 :5000, 1 :7500 or 1 : 10,000.
• These unique variants serve as a personalized biomarkers, where a quantitative PCR-based assay could accurately measure the subject's tumor burden.
• the variants can then be used in prognosing, determining a treatment regimen, determining response to treatment or detecting recurrence of disease by detecting the variant polynucleotide.
• the method comprises a further step prognosing, determining progression of cancer, predicting a therapeutic regimen or predicting benefit from therapy in a subject having a disease based on the detection of a variant.
• the disease is cancer.
• the cancer is selected from the group consisting of a carcinoma, sarcoma, leukemia, lymphoma, myeloma, and a CNS tumor.
• the cancer is selected from the group consisting of skin cancer, lung cancer, colon cancer, pancreatic cancer, prostate cancer, liver cancer, thyroid cancer, ovarian cancer, uterine cancer, breast cancer, cervical cancer, kidney cancer, epithelial carcinoma, squamous cell carcinoma, basal cell carcinoma, melanoma, papilloma, and adenomas.
• the invention provides a kit for detecting a variant polynucleotide having a nucleotide sequence differing from the wildtype nucleotide sequence of a nucleic acid molecule, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the kit comprising a) a multiplicity of primers; and b) algorithms to detect the variant.
• the variant is a mutation, deletion, insertion, substitution or genomic rearrangement.
• the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately 1-20 kb around the locus of interest and the innermost primers is separated by approximately at least 15-100 kb. In an additional aspect, the multiplicity of primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 5-15 kb around the locus of interest and the innermost primers is separated by approximately at least 20-80 kb.
• the primers are designed to hybridize to loci on the wildtype nucleic acid molecule evenly spaced approximately at least 6 kp around the locus of interest and the innermost primers is separated by approximately at least 20 kb. In a further aspect, the ratio of primer spacing to locus of interest is 6 kp to 80 kb.
• the present invention provides a system for analyzing a variant polynucleotide of unknown nucleotide sequence believed to differ from the wildtype nucleotide sequence of a nucleic acid molecule of interest in a subject, comprising: a) a multiplicity of primers computationally designed to detect one or more breakpoints in the wildtype nucleic acid sequence of the subject; b) a computer -executable algorithm for detecting the variant in a sample from a subject having a cancer, the algorithm comprising computational design of a multiplicity of primers; c) use of the multiplicity of primers in multiplex PCR to amplify any variant polynucleotide present in the sample; d) analysis of the sequence of each multiplex PCR products; e) detection of the variant polynucleotide in the sample; and f) long range sequencing of the variant polynucleotide to confirm the differences in nucleotide sequence in the variant as compared to the sequence of
• system further comprising a machine to perform the multiplex PCR. In another aspect, the system further comprising a machine to sequence the multiplex PCR products. In a further aspect, the system further comprising a machine to perform long range sequencing of a detected variant polynucleotide.
• the invention provides a method for confirming variant polynucleotide of unknown nucleotide sequence which differs from the wildtype nucleotide sequence of a nucleic acid molecule of interest, wherein the variant polynucleotide is in a sample containing up to about 99.9% of the wildtype nucleic acid molecules, the method comprising: a) sequencing the nucleotide of interest; and b) analyzing the sequence by i) sequence alignment to wild-type nucleotide sequence, ii) alignment trimming, iii) clustering of breakpoints, and iv) confirming the variant sequence.
• the high through-put sequence analysis is single molecule sequence analysis.
• the high through-put sequence analysis has a high error rate.
• the high through-put sequence analysis error rate is comprised of up to about 20% insertion error rates and deletion rates and/or up to about 5% substitution error rates.
• AmBre exploits the fact that variable breakpoints aggregate along fragile regions of the chromosome by designing primers around the fragile regions, this was used to produce a single design for five cancer cell lines: A549, CEM, Detroit562, MCF7, and T98G.
• Breakpoints were estimated by copy number changes for four cancer cell-lines (A549, CEM, MCF7, and T98G) from SNP-array data (Table 1) and the breakpoint was given for a fifth cell line (Detroit562) from prior studies.
• the error in breakpoint estimation for SNP-array data is roughly lOKbp.
• AmBre-design output a high quality 16 primer design (AMBRE- 16) with primers spaced apart by approximately 6Kbp to cover the lOOKbp input region.
• the design was used by AmBre-amplify on DNA samples from each cell line.
• the experiment successfully amplified DNA from each cell line (S.M. 2), where each line produced a unique sized amplicon even though the same set of 16 primers are used for each reaction.
• Primer3 2.3.0 was used with long-range PCR specific parameters to identify 3 lbp candidate AmBre primers that were capable of amplification under the same thermocycling conditions.
• candidate primers were aligned to the reference human assembly (GRCH37) using Blat. Define an end-aligning match as an exact match of length > 18 between the 3' end of a primer and an off-target location. Primers with greater than 10 end-alignments were removed as having a high chance for off-target amplification. Second, pairs of primers that have compatible end-alignments within a 2d long off target region were marked as incompatible.
• each pair (including a self-pair) was tested for dimerization using Multi-Plx.
• Primers with self-dimerization (maximum binding energy AG less than -8.0 kcal/mol for any region) were removed and pairs with high binding affinity (maximum binding energy AG less than -4.0 kcal/mol for primer-primer 3end binding or -8.0 kcal/mol for any region of primers) were marked as incompatible.
• the remaining candidate primers and incompatibilities formed the input to AmBre primer selection.
• the candidate primer generation and primer filtering stages resulted in 5181 candidate primers.
• the candidate primers are uniformly spread across breakpoint regions suggesting good tiling primer designs may exist.
• the simulated annealing algorithm is repeated for 12 different rates of convergence with the fastest convergence rate having a 10 minute average runtime and slowest convergence rate having a 864 minute average runtime (Fig. 3b).
• d 6500
• the lowest cost solution AMBRE-68
• a final AmBre primer design was selected from a filtered list of candidate primers (Pu) and primer-primer compatibilities.
• a low cost P according to C(P) we applied a simulated annealing procedure, an initial design P was computed using a random subset of 6 primers. Define the neighboring design of P, N(P), as either the removal of a single primer from P, or the addition of a single primer pe P to P followed by removal of all primers p P s.t. ( ⁇ , ⁇ )' e E.
• the simulated annealing procedure described in Algo 1 was used to compute low cost designs.
• PCR was performed using the following thermocycling conditions; initial denaturation at 95°C for 3 min, 10 cycles at 94°C for 20 sec, 64°C for 30 sec, 66°C for 15 min, 28 cycles at 94°C for 5 sec, 64°C for 30 sec, 66°C for 15 min + 20 sec for each cycle, final extension at 64°C for 45 min, and 4° hold.
• the standard protocol for NEB CrimsonTM LongAmp Taq was used for 50 ul PCR reactions with the following changes. The same mix of 16 primers was used in each reaction where each primer is present with final concentration of 0.2 ⁇ . Starting genomic DNA for each cell line reaction is 10 ng. QIAquick PCR purification kit was used to clean up PCR. samples.
• sequence data was the input to AmBre-analyze.
• the tool BLASR identified 52k alignable fragments. After clustering in AmBre-analyze, deep coverage of every breakpoint was retrieved (although with 6 clusters instead of 5; see below), with A549 having the lowest coverage of 300 fragments and CEM having the highest coverage of 15,000 fragments (Fig. 5b).
• the difference in coverage is due to different amplicon sizes, where shorter amplicons are easier to load onto a PacBioTM SMRT cell than longer amplicons. Later generation PacBioTM instrumentation normalizes for this sequencing bias.
• Table 1 Five cell-lines with CDKN2A deletion breakpoints in GRCH37. Estimated breakpoints are according to CGP. COP coordinates were converted from GRCH36 to GRCH37 using UCSC liftover. The break coordinates for Detroit562 were identical to those previously described and the cell-line was not examined by CGP.
• AmBre can be applied to contiguous break regions.
• a 68 primer design was developed to capture CDKN2A deletions with breaks in a 480kb region (AMBRE-68, see also Figure 3).
• PCR was performed using the standard protocol for NEB Crimson LongAmp Taq is used for 50 ⁇ PCR reactions with the following changes. The same mix of 9 primer was used in each reaction where each primer is present with final concentration of 0.4 ⁇ . Starting genomic DNA for each cell line reaction is 20ng.
• the AmBre assay can target DNA with the relevant deletion in the context of high background of germline DNA. This feature is important for sensitive detection of tumor DNA and establishing a patient specific tumor DNA marker for monitoring tumor burden.
• each reaction starts with a heterogeneous mixture of approximately 400ng with tumor to wild-type gDNA mixture ratios of 1 : 1 , 1 : 10, 1 : 100, 1 : 1000.
• each reaction contains numerous primers where only 2 primers are responsible for amplification.
• each reaction contains 16 primers sampled from
• A549, CEM, Detroit562, and T98G cells were thawed from Moore's Cancer Biorepository. MCF7, HeLa, and HEK (293T) cells were collected. Standard DNAzol protocol was used for DNA extraction and DNA was quantified with NanoDrop 2000 spectrophotometer. DNA products are visualized on 1% agarose gels with EtBr. Gel images are either color value inverted or color curve adjusted uniformly across the image for visual enhancement. All PCRs were performed on a BioRad iCyclerTM instrument.
• AmBre also captures more complex rearrangements like inter-chromosomal translocations. This was demonstrated with an experiment characterizing RUNX1-RUNX1T1 gene fusion, the results of a translocation between chr21 and chr8. In the tumor genome, breakpoint ends lie within a 30Kbp region chr21 : 36205000-36235000) in the RUNX1 intron, and a 55Kbp region chr8 : 93030000-93085000 in RUNXITI, and the derivative chromosome 8 (Der8) encodes a fusion oncoprotein.
• the 7 translocation is balanced and also generates a fusion of RUNX1T1-RUNX1 on a derivative chromosome21 (Der21).
• Der21 derivative chromosome21
• AmBre was used to design 10 reverse primers in the RUNXl region and 18 forward primers in the RUNX1T1 region with ⁇ 3Kbp primer spacing.
• a subsampling of primers and efficacy in generating longer amplicons were investigated.
• forward and reverse primers were divided based on index parity when sorted by chromosome position.
• the forward and reverse primer sets make four combinations: FO U RO, FO U RE, FE U RO, and FE U RE, primers for capturing target breakpoints.
• These combinations can be treated as four new primer designs, each with a maximum product size of 12Kbp, but half as many primers.
• amplification efficiency may be assessed across different amplicon lengths and primer density per reaction using the same DNA template.
• the Kasumi-1 breakpoints for Der8 were generated by the sixth forward and ninth reverse primer.
• 14 primer designs FE U RO, FO U RO, and FO U RE produce 3:5Kbp, 6:8Kbp, and 10: lKbp amplicons (Fig. 10).
• AmBre was used to identify CDKN2A deletion breakpoints in primary tumors. Namely, tumor tissue from three pancreatic cancer patients were expanded in xenograft mouse models. DNA was collected and SNP-array analysis indicated a deletion in the CDKN2A locus. AmBre designed primers for each of these DNA samples and amplified DNA harboring the CDKN2A deletions. After simultaneous Pacific Biosciences RSTM sequencing of these products and sequencing data analysis, 1.67Mbp, 308Kbp, and 821Kbp deletions were confirmed in the pancreatic cancer samples.

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