RU2613489C2 - Method of detecting mutations in complex dna mixtures - Google Patents

Abstract

FIELD: biochemistry.

SUBSTANCE: in some applications non-mutant DNA is present in the sample in thousandfold excess. Method involves molecular bar-coding of DNA molecules, their amplification, sequencing of new generation and analysis of sequence data.

EFFECT: present invention is aimed at methods for detection in sample of mutations in DNA, where mutant DNA is present on the background of a non-mutant DNA.

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Description

Field of Invention

[01] The invention relates to the field of molecular biology and medicine. In particular, the invention is directed to methods for detecting mutations in DNA samples.

State of the art

[02] Early diagnosis and treatment of cancer remain one of the most pressing problems of modern medicine. The identification of such diseases in the early stages and the selection of an adequate treatment strategy can significantly increase patients' chances of recovery and reduce cancer mortality rates.

[03] The main reason for the occurrence of malignant neoplasms in the human body are mutations that damage the functioning of the most important regulatory genes. The nature of these mutations determines the type of tumor, the degree of their malignancy and the sensitivity of the tumor to drugs. Analysis of DNA molecules carrying mutations of tumor origin (oncomutation) allows us to predict the aggressiveness of tumors and the likelihood of metastases, as well as recommend the choice of treatment strategy. At the same time, oncomutations have almost absolute specificity, which makes them suitable for evaluating the drug sensitivity of a tumor and detecting acquired resistance. Thus, the detection of cancer mutations is of great prognostic and diagnostic value, which has been proved by many years of practice (see, for example, van der Heijden, MS, et al. Clin Cancer Res. 2005 Oct 15; 11 (20): 7508-15; Thomas , RK, et al. Nat Genet. 2007; 39 (3): 347-51; Diehl, F., et al. Nat Medicine. 2008; 14 (9): 985-90; Rizvi, Ν.Α., et al. Clin Cancer Res. 2011; 17 (10): 3500-6).

[04] Analysis of mutations in “hot spots” of key oncogenes and suppressor genes, including mutations in exon 15 of the BRAF gene, exon 7 of the ARAF gene, exon 7 of the RAF1 gene, exon 11 of the KIT gene, exons 2, 3, 4 of the KRAS gene, exons 2, 3, 4 of the NRAS gene, exons 2, 3, 4 of the HRAS gene, exons 2, 3, 6 of the MAP2K1 gene, exons 4 and 6 of the MAP2K2 gene, exon 7 of the MAPK1 gene, exons 9 and 20 of the PIK3CA gene, exon 2 of the AKT1 gene, exon 5 of the GNAQ gene, exon 5 of the GNA11 gene, exon 2 of the RAC1 gene, exons 12, 18, 19, 20, 21 of the EGFR gene, exons 8, 18, 19, 20, 21 of the HER2 gene, exons 2, 3, 6, 7, 8, 9, 21, 23 of the HER3 gene, exons 8, 12, 14, 21 of the HER4 gene, exons 5, 6, 7, 8, 9 of the TP53 gene and exon 3 of the CTNNB1 gene has a significant diag nostic, prognostic and predictive value in many types of cancer, including lung cancer, breast cancer, colon and rectal cancer, malignant melanoma of the skin, thyroid cancer, cancer of the stomach, pancreatic and bile duct cancer, bladder cancer, glial tumors of the head brain, sarcomas of bones and soft tissues, many types of leukemia and lymphomas (Vogelstein, B., et al. Science. 2013 Mar 29; 339 (6127): 1546-58; Watson, IR, et al. Nat Rev Genet. 2013 Oct; 14 (10): 703-18; Weinstein, JN, et al. Nat Genet. 2013 Oct; 45 (10): 1113-20; Kandoth, C., et al. Nature. 2013 Oct 17; 502 (7471): 333-9; http : cancer . sanger . ac . uk ; http : cancergenome . nih . gov ; http : p 53. free . fr ; and many others etc.) Many of these mutations can be used as non-invasive markers of tumor presence, including in the early stages of the disease (Fleischhacker, M., Schmidt, B. Biochim Biophys Acta. 2007; 1775 (1): 181-232; Chen, Z., et al. PLoS One. 2009; 4 (9): e7220; Newman, AM, et al. Nat Med. 2014 May; 20 (5): 548-54).

[05] To date, a significant number of methods have been developed for detecting mutations in DNA. Most of these methods include the selective amplification of alleles carrying mutations or polymorphisms during PCR. The following main groups of methods can be distinguished: allele-specific PCR (Newton, C. R., et al. Nucleic Acids Res. 1989 Apr 11; 17 (7): 2503-16; Ruano, G., Kidd, KK Nucleic Acids Res . 1989; 17 (20): 8392; Sommer, SS, et al. Mayo Clin Proc. 1989; 64 (11): 1361-72), ligation-mediated PCR (Wu, DY and Wallace, RB Genomics. 1989; 4; 4). (4): 560-9; Shi, C., et al. Nat Methods. 2004; 1 (2): 141-7); corrective PCR (Zhang, J., et al. Trends Biotechnol. 2005 Feb; 23 (2): 92-6; Liu, Q., Sommer, S.S. Biotechniques. 2000 Nov; 29 (5): 1072-6); Cold PCR (Li, J., et al. Nat Med. 2008; 14 (5): 579-84; Milbury, C.Α., et al. Nucleic Acids Res. 2011; 39 (1): e2) ; Wild-type PCR (Seyama, T., et al. Nucleic Acids Res. 1992; 20 (10): 2493-6; Orou, Α., Et al. Hum Mutat. 1995; 6 (2): 163-9 ; Beau-Faller, M., et al. Br J Cancer. 2009; 100 (6): 985-92).

[06] A significant drawback of the first two methods is the actual introduction of the desired allelic variant into the matrix during PCR. The product obtained using these methods cannot be tested in any additional way, since the truly positive and false positive results give exactly the same picture.

[07] The method of correcting PCR is based on reactions that under any conditions proceed with very low efficiency; as a result, it is not entirely applicable to most tasks of searching for rare mutations and polymorphisms, in which partially degraded DNA samples are studied, and the number of desired molecules “at the start” can be calculated in units.

[08] The methods of “cold” PCR and wild-type PCR blocking are based on extremely small differences in the melting temperature of perfect and imperfect duplexes; therefore, to obtain reproducible results when using these methods, it is necessary to resort either to the use of modified oligonucleotide probes (LNA, PNA, P-HyP-PNA, ZNA, etc.), which are expensive and difficult to synthesize, or to special modifications of PCR devices , allowing to transform a small difference in the melting temperature into a significant difference in the effectiveness of PCR (and such devices are much more expensive than standard ones, and the selection of PCR conditions for them is quite complicated), or to extremely inconvenient modes PCR (60 cycles of more than 10 minutes each) (Galbiati, S., et al. Clin Chem. 2011; 57 (1): 136-8).

[09] However, when optimizing conditions, the above methods can provide selectivity of the order of 1: 100-1: 200 (Pao, W., Ladanyi, M. Clin Cancer Res. 2007; 13 (17): 4954-5; Milbury, C.Α., et al. Clin Chem. 2009; 55 (4): 632-40) and are widely used for mutation analysis. However, to obtain DNA samples suitable for analysis by these methods, it is necessary to take a biopsy directly from the tumor, which is impossible in the early stages of cancer. In addition, all these methods allow you to analyze only certain mutations at previously known points, which significantly reduces the information content of the analysis.

[010] In recent years, it has been found that DNA fragments carrying mutations of tumor origin are present in human biological fluids even with a minimal tumor focus (Diehl, F., et al. Proc Natl Acad Sci USA. 2005; 102 (45) : 16368-73; Fleischhacker, M., Schmidt, B. Biochim Biophys Acta. 2007; 1775 (1): 181-232; Chen, Z., et al. PLoS One. 2009; 4 (9): e7220). Detection of mutations in tumor DNA samples from the blood of patients opens up prospects for early diagnosis of the tumor and its relapses, monitoring of residual disease and assessing the radical nature of the treatment.

[011] However, the identification of such mutations is confronted with unresolved technological problems to date. The main ones are: (a) the need for a simultaneous analysis of the oncomutation panel against the background of an extremely small amount of circulating DNA in biological fluids, and (b) the presence of non-mutant DNA in biological fluids in a ratio of 1000-100000 copies per tumor molecule. These problems make it impossible to use the classical methods of mutational analysis listed above, since their resolution is orders of magnitude lower than the required level (Liu, Q., Sommer, SS Hum Mutat. 2004 May; 23 (5): 426-36; Gilje, B ., et al. J Mol Diagn. 2008 Jul; 10 (4): 325-31; Busby, K., Morris, AJ Clin Pathol. 2005, 58 (4): 372-5). Thus, there is a need for mutation detection methods capable of detecting mutant nucleic acid against a background of a thousand-fold (or higher) excess of reference nucleic acid.

[012] The method proposed in this application is aimed at detecting mutations in DNA samples, where the mutant molecules are in a mixture with an excess of normal (non-mutated) DNA. The method finds application, for example, to detect mutations, the analysis of which is required for the diagnosis and monitoring of diseases, in DNA of tumor origin isolated from biopsy material or from biological fluids.

[013] The proposed method is based on molecular barcoding, subsequent amplification of target DNA molecules and their sequencing by high-performance sequencing methods, such as new generation sequencing methods.

[014] As used here, the terms “high throughput sequencing”, “next generation sequencing” or “NGS” (from next generation sequencing) refer to a group of technologies for simultaneously sequencing a huge number of single DNA molecules, including 454 technology, Illumina technology (Solexa SBS Technology), SoLID technology, and IonTorrent technology (Metzker ML Nat Rev Genetics. 2010; 11 (1): 31-46; Mardis, J. R. Annual review of genomics and human genetics. 2008; 9: 387-402; Bentley DR, et al. Nature. 2008; 456 (7218): 53-59; Rothberg JM, et al. Nature. 2011; 475 (7356): 348-352; Drmanac R., et al. Science. 2010; 327 ( 5961): 78-81; Eid J., et al. Science. 2009; 323 (5910): 133-138; Lam HY, et al. Nature biotechnology. 2012; 30 (1): 78-82 ; Clark, M.J., et al. Nat Biotech. 2011; 29 (10): 908-914; Quail M.A., et al. BMC genomics. 2012; 13: 341). For all these technologies, the stage of sample preparation is the introduction of the so-called “sequence adapters” into the DNA sequence so that the DNA molecule is flanked on one side by adapter A and on the other by adapter B. Sequence adapter sequences are known to those skilled in the art and provided by manufacturers of next-generation commercial sequencing kits.

[015] A close analogue of the proposed method is the method proposed by Schmitt and colleagues (Schmitt, M.W., et al. Proc Natl Acad Sci USA. 2012 Sep 4; 109 (36): 14508-13). The method includes the steps of synthesizing double-stranded molecules of barcoding DNA adapters, ligating these DNA adapters to DNA molecules of a sample, PCR amplification with universal primers, next generation sequencing (NGS), and analyzing data with PCR and NGS error filtering. The synthesis of barcode-bearing adapters is carried out using PCR on a synthetic DNA matrix - an oligonucleotide containing a random sequence of 6-24 nucleotides.

[016] The claimed method differs from that indicated by the technology of barcoding: (1) polymerase synthesis of double-stranded barcoding DNA adapters is not used, which significantly reduces the risk of errors within the barcodes themselves; (2) the introduction of barcodes is carried out using linear PCR, which can significantly increase the diversity of the library, dramatically increase the specificity of the analysis by cutting off non-target molecules at the amplification stage, and also reduce the time of sample preparation in the case of analysis of a finite number of regions of the genome.

[017] Another method is described by Kinde et al. (Kinde, I., et al. Proc Nat Acad Sci USA. 2011; 23: 9530-35). The method includes the introduction of synthetic barcoding primers using PCR, further PCR amplification of the barcoded molecules using universal primers, sequencing of the new generation (NGS), and analysis of data with PCR and NGS error filtering. For the introduction of barcodes, 2 PCR cycles are used in the presence of the barcoding and counter primers.

[018] Barcoding using standard PCR has several disadvantages. Due to the fact that only DNA molecules of the initial sample (but not the products of their amplification) should be barcoding, it is possible to use no more than two cycles of standard PCR (and ideally, one cycle). Thus, each source molecule corresponds to a single barcoded copy. This makes it impossible to further distinguish polymerase synthesis errors from true mutations due to "filtering by quantity." The principle of “filtering by quantity” is based on the fact that true mutations that were originally present in the sample should be found in the “raw data” of sequencing significantly more often than random substitutions that occurred during sample preparation and sequencing. In addition, in this case, there is no increase in the initial pool of molecules for further analysis, which complicates the work with small amounts of biological material.

[019] The claimed method involves a more efficient barcoding procedure using linear PCR. The use of linear PCR for introducing molecular barcodes has a number of fundamental advantages compared to standard PCR, in particular: 1) the restriction on the number of barcoding reaction cycles is removed, which makes it possible to efficiently work with low amounts of initial DNA, as well as detect DNA polymerase errors, arising in the process of barcoding, by "filtering by quantity"; 2) the strand specificity of the reaction is preserved, which allows one to distinguish true mutations from the results of damage to the original DNA by staging two reactions (the first with a direct primer and the second with a reverse primer in the same region of the genome). Optimized conditions of the exonuclease reaction used in a number of applications of the claimed method allow efficient hydrolysis of unused primers without significant degradation of linear PCR products. In addition, the claimed method, in contrast to that described by Kinde and colleagues, is adapted for multiplex analysis of target regions of the genome.

SUMMARY OF THE INVENTION

[020] The present invention provides a method for detecting mutations in target DNA sequences (mutant DNAs) that distinguish target sequences of an analyzed DNA sample from reference DNA sequences.

[021] In some applications, the mutated DNA contains one or more point mutations. In some applications, the mutant DNA contains one or more deletions of one or more nucleotides. In some applications, the mutant DNA contains one or more insertions of one or more nucleotides.

[022] The method of the present invention includes:

(a) molecular barcoding of a pool of target DNA molecules in a DNA sample;

(b) purification of the product of the barcoding reaction from residues of the barcoding primer;

(c) amplification of a pool of target molecules;

(d) introducing sequence adapters into the PCR product and sequencing said PCR product;

(e) the identification of mutations in the target DNA according to sequencing data.

[023] The claimed method allows you to detect mutations in the target DNA sequence in samples that contain both mutant and non-mutant DNA, and the ratio of the concentration of mutant to non-mutant DNA can be less than 1: 100, for example, in the range 1: 100-1: 100,000, for example, be less than 1: 140, be less than 1: 300, be less than 1: 1000, be less than 1: 5000, or be less than 1: 10000.

[024] In particular, the claimed method allows the detection of mutations in fragments of genes selected from SEQ ID No: 1 - SEQ ID No: 62. The number of target sequences selected for analysis may vary depending on the purpose of the analysis and includes at least five target sequences, usually 6, 7 or more sequences, for example, ten or more sequences, more often seventeen or more sequences, thirty or more sequences, forty or more sequences, fifty or more sequences, sixty and more than 70 sequences, seventy or more sequences, for example, 10, 17, 18, 27, 28, 34, 35, 44, 45 or 62 sequences.

[025] In preferred embodiments, molecular barcoding is performed by multiplex linear PCR in the presence of barcoding primers. The number of barcoding primers used for linear PCR is equal to the number of sequences selected for analysis.

[026] In some embodiments, linear PCR is performed in one tube, in some embodiments, linear PCR is performed in two, three, four or more tubes, each of which contains its own set of barcoding primers.

[027] In preferred embodiments, purification from the barcoding primer comprises treating the reaction mixture with exonuclease I.

[028] Barcoded DNA molecules are amplified in multiplex PCR and the amplification product used for sequencing. PCR is carried out in the same number of tubes as the barcoding of target molecules. In preferred embodiments, the amplification product is sequenced by high throughput sequencing methods.

[029] In some embodiments, the introduction of sequencing adapters is carried out using PCR with the release of in the presence of recessed internal gene-specific primers.

[030] In preferred embodiments, the analysis of the descendants of each target molecule from the original DNA sample is used to detect mutations in the sequencing data, where the descendants of one molecule are identified by the sequence of an individual barcode.

[031] Also provided are primer sets for implementing the method of the present invention for detecting mutations in DNA fragments selected from the group of SEQ ID No: 1 - SEQ ID No: 62.

[032] Reagent kits are also provided for implementing the method of the present invention for detecting mutations in DNA fragments selected from the group of SEQ ID No: 1 to SEQ ID No: 62. In preferred embodiments, the kits include barcode primers and primers for amplification of the target molecules. In some embodiments, the kits also include primers for introducing sequences of sequence adapters into the target molecules using in-depth PCR and outward PCR. In some embodiments, the kits also include reagents for the implementation of PCR. In some embodiments, the kits also include reagents for purifying the products of the barcoding reaction.

Brief Description of Drawings

[033] Figure 1 shows the structure of a barcoding primer.

[034] Figure 2 illustrates the sequence of sample preparation procedures of a preferred embodiment of the method of the present invention.

[035] Figure 3 illustrates various methods for introducing sequence adapters.

[036] Figure 4 shows the electrophoregrams of the products of two consecutive PCR rounds after the barcoding reaction in the presence of two primers (lane 1) and after the barcoding reaction in the presence of one primer (lane 2). M - DNA length marker 100+ bp DNA Ladder (Eurogen, Russia).

[037] Figure 5 shows an electrophoregram of the amplification products of BRAF-wt samples (lane 1); BRAF-mut100 (lane 2); BRAF-mut1000 (lane 3). M - DNA length marker 100+ bp DNA Ladder (Eurogen, Russia).

DETAILED DESCRIPTION OF THE INVENTION

[038] As indicated above, the present invention is directed to a method and kit for detecting mutations in a DNA sample.

[039] The method of the present invention includes

[040] (a) molecular barcoding of a pool of target DNA molecules in a DNA sample;

[041] (b) purification of the product of the barcoding reaction from residues of the barcoding primer;

[042] (c) amplification of a pool of target molecules in multiplex PCR;

[043] (d) introducing sequence adapters into a multiplex PCR product and sequencing said multiplex PCR product;

[044] (e) the identification of mutations in the target DNA according to sequencing data.

[045] The claimed method allows to detect mutations in the target DNA sequence in samples that contain both mutant and non-mutant DNA, and the ratio of the concentration of mutant to non-mutant DNA can be 1: 300-1: 100000, for example, 1: 2000, 1: 5000 or 1: 10000.

[046] This method is used in many applications, in particular, for the detection of somatic mutations for a wide range of problems in biology and medicine, in the analysis of the level of spontaneous mutagenesis and in the analysis of genetic polymorphism in pulled samples (in particular, in population, evolutionary and environmental genetics). In particular, the method of the present invention finds application for detecting the tumor presence by analyzing extracellular DNA from biological fluids (blood, urine).

[047] Kits for implementing the method of the present invention are also provided.

[048] In preferred embodiments, the kits of the present invention allow the analysis of mutations in fragments of genes selected from the group of SEQ ID No: 1 - SEQ ID No: 62. The method of the present invention can also be used to analyze any other mutations, however, this requires the selection of suitable gene-specific sequences of primers and multiplex PCR conditions.

[049] Various terms related to the biological molecules and methods of the present invention are used above and also in the description and in the claims.

[050] It should be noted that, as used here and in the appended claims, singular forms include plural reference objects unless the context clearly requires otherwise. For example, reference to “nucleic acid” includes many molecules of such nucleic acids.

[051] As used here, a nucleic acid molecule is a DNA molecule, such as a genomic DNA or cDNA molecule. As used here, the term "cDNA" refers to nucleic acids that possess a sequence of sequence elements found in native mature types of mRNAs, where sequence elements are exons and 5'- and 3'-non-coding regions.

[052] As used here, the term "sample preparation" refers to all the manipulations to which nucleic acids are subjected after purification and prior to sequencing.

[053] As used here, the term "mutant" or "mutant" refers to a DNA molecule whose nucleotide sequence is different from the reference DNA sequence. In addition, the term "mutant" refers to any variant that is shorter or longer than the reference DNA. In some applications, the difference is spot replacement. In some applications, the difference is a deletion or insertion of one or more nucleotides. For the needs of the present invention, the size of the deletion or insertion does not exceed 150 nucleotides, preferably does not exceed 100 nucleotides. With the exception of these differences, the sequences of the mutant and reference DNA are identical. In preferred applications, the reference DNA sequence is identical to the wild-type human DNA sequence.

[054] As used here, the term "hot spot" refers to a position in a DNA molecule if a mutation often occurs in this position under certain conditions. So, in relation to key oncogenes and suppressor genes, the term "hot spot" refers to the position of a mutation that often occurs in oncogenesis. In relation to PCR errors, the term “hot spot” denotes a position in the DNA matrix if polymerase DNA synthesis errors are predominantly localized in this position.

[055] The percentage of sequence identity with the reference is determined by comparative analysis. Algorithms for sequence analysis are known in the art, such as BLAST described in: Altschul et al. J. Mol. Biol., 1990, 215, pp. 403-10. For the purposes of the present invention, nucleotide and amino acid sequence comparisons performed using the Blast software package provided by the National Center for Biotechnology Information ( http: www.ncbi.nlm.nih.gov/blast ) using alignment breaks with standard parameters can be used to determine the level of identity and similarity between nucleotide sequences.

[056] As used here, the nucleotide sequences are "substantially similar" or "substantially the same as the reference sequence" if the nucleotide sequences have at least 85% identity with each other or with the specified sequence within the region selected for comparison. Thus, substantially similar sequences include those that have, for example, at least 85% identity, at least 90% identity, at least 95% identity, or at least 96%, 97%, 98 % or 99% identity. Two sequences that are identical to one another are also essentially similar.

[057] As used here, the term "PCR" refers to the well-known method of polymerase chain reaction (described, for example, in Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, New York, 1989; Erlich, PCR Technology , Stockton Press, New York, 1989; Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Harcourt Brace Javanovich, New York, 1990; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; Barnes, WM Proc Natl Acad Sd USA, 1994, 91, 2216-2220). A standard PCR reaction mixture includes DNA polymerase, a buffer that ensures the optimal functioning of DNA polymerase, magnesium chloride, deoxynucleotide triphosphates, DNA matrix, and oligonucleotide primers.

[058] The reaction mixture for PCR contains Mg2 + ions in a concentration of 0.1-10 mM, deoxyribonucleoside triphosphates (dNTPs), including ATP (dATP), GTP (dGTP), CTF (dCTP) and TTF (dTTP), in a final concentration of 20 -800 μM each, typically 160-600 μM, preferably 200-300 μM. The PCR reaction mixture may contain other components, for example, substances that facilitate the amplification of GC-rich regions, antibodies that provide a hot start, etc. Such components are well known to those skilled in the art.

[059] The volume of the reaction mixture for PCR is usually 10-100 μl, usually 15-50 μl, for example, 20 μl or 25 μl. The amount of DNA template added to the PCR reaction mixture is in the range of 10 pg to 1 μg, typically from 100 pg to 200 ng, for example, 2 ng per reaction, or 5 ng per reaction, or 10 ng per reaction, or 50 ng per reaction, or 100 ng per reaction.

[060] As used here, the term "primer" refers to oligonucleotides routinely used in PCR as a seed for DNA polymerase. As used here, the term "gene-specific" refers to sequences of primers capable of annealing (having an "annealing site") to the target nucleic acid due to its constituent sequences complementary to the specified nucleic acid. Synthetic oligonucleotides act as primers. Synthetic oligonucleotides can be prepared using the phosphoramidite method and purified using methods well known in the art, such as high performance liquid chromatography (HPLC), or other methods, as described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2 ^nd Ed., (1989) Cold Spring Harbor Press, Cold Spring Harbor, NY, or according to the instructions described in, for example, United States Dept. of HHS, National Institute of Health (NIH) Guidelines for Recombinant DNA Research.

[061] The oligonucleotide primers used for PCR have an annealing site for target sequences, for example, sequences introduced into DNA molecules during previous sample preparation (the so-called universal or adapter sequences) or gene-specific sequences of target molecules. The term "annealing site" in this context means a nucleotide sequence that is complementary to the target sequence and serves as a seed for DNA polymerase.

[062] Methods for selecting a gene-specific sequence for a primer are well known. As a rule, the length of a gene-specific sequence is not less than 10, but not more than 100 nucleotides, usually 15-30 nucleotides. The adapter sequence is chosen so as to minimize non-specific amplification during subsequent PCR. The length of the adapter sequence can vary from 10 to 100 nucleotides and typically is 15-30 nucleotides.

[063] As used here, the term "target nucleic acid" refers to a nucleic acid in which mutations are of interest.

[064] As used here, the term "isolated" means a molecule or cell that is in an environment other than the environment in which the molecule or cell is in vivo.

[065] As used here, the terms "tumor DNA", "DNA of tumor origin" or the like mean DNA or cDNA originating from malignant tumor cells.

[066] As used here, the term "oncutation" means a change in the gene sequence (point change, deletion or insertion) that has occurred in the tumor DNA.

[067] As used here, the term "extracellular DNA" means DNA located in biological fluids circulating in the extracellular space (for example, in urine or in blood plasma).

[068] Obtaining a DNA sample containing the target DNA molecule

[069] Mutant DNA may be present in a test sample of DNA isolated from biological material, or may be added exogenously.

[070] Differences in the nucleotide sequence between highly similar nucleotide sequences of mutant and reference DNA (substitutions, deletions and insertions) can represent single nucleotide polymorphisms, as well as mutations in the sequence that occur during normal replication or duplication, including somatic mutations or mutations de novo.

[071] Other mutations (substitutions, deletions and insertions) can be specifically calculated and inserted into the sequence for specific purposes, such as the creation of artificial mutant DNA. Such special substitutions can be made in vitro using various mutagenesis technologies or obtained in host organisms under specific breeding conditions that induce or select these changes. Such specially prepared sequence variants may be called “mutants” or “derivatives” of the original sequence.

[072] Mutant DNAs can be obtained on a template nucleic acid by modifying, deleting, or adding one or more nucleotides in a template sequence, or a combination thereof, to obtain a template template nucleic acid. Modifications, additions or deletions can be performed by any method known in the art (see, for example, Gustin et al. Biotechniques. 1993. 14:22; Barany Gene. 1985. 37: 111-123; Colicelli et al. Mol. Gen. Genet. (1985.1993: 537-539; Sambrook et al., Molecular Cloning: A Laboratory Manual, (1989), CSH Press, pp. 15.3-15.108), including error prone PCR, shuffling, oligonucleotide directed mutagenesis, assembly PCR, paired PCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursive multiple mutagenesis, exponential multiple mutagenesis, site-specific mutagenesis, random mutagenesis, gene reass gene reassembly, gene site-saturating mutagenesis (GSSM), ligation-assisted rearrangement (SLR), or combinations thereof. Modifications, additions or deletions can also be performed by a method including recombination, recursive recombination of sequences, phosphotioate-modified DNA mutagenesis, mutagenesis on a uracil-containing matrix, double-pass mutagenesis, mismatch-specific reductive mutagenesis, reduction-deficient mutagenesis, chemical mutagenesis, radioactive mutagenesis, deletion mutagenesis, restriction-selective mutagenesis, purification restriction mutagenesis, synthesis of artificial genes, multiple mutagenesis, the creation of chimeric multiple nucleic acids and their combinations.

[073] A DNA sample can be isolated from biological material, for example, from cultures of cells, tissues, smears and scrapings, biopsy material, body fluids (blood, urine, saliva) of animals, including humans. Any method known in the art can be used to isolate genomic DNA. For example, the methods described by Sambrook et al. Can be used. (Molecular Cloning: A Laboratory Manual, 2nd edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989), or commercially available DNA isolation kits, such as DiaTom or MagNA kits, Isogen Laboratory LLC, Russia ) or the DNEasy series (QiaGen company, Germany) or their analogues.

[074] Total biological RNA or a poly (A) RNA fraction can also be isolated from biological objects. RNA isolation can be performed by any method known in the art (for example, commercially available RNA isolation reagents such as Trizol (GIBCO / Life Technologies), TRI Reagent (Ambion), or RNeasy kits (QIAGEN) or the guanidine method can be used thiocyanate-phenol-chloroform extraction described by Chomczynski & Sacchi, 1987 (Anal. Biochem. 162, 156-159). RNA samples can be used to prepare cDNA samples using synthesis techniques known in the art, for example, the Hilbert-Hoffman method (Gubler, U., Hoffman, BJ Gene. 1983. V. 25. P. 263-269), methods using DNA-l gases of phage T4 (Akowitz & Manuelidis Gene. 1989. V. 81. P. 295-306; Lukyanov et al. Biophys. Biochem. Res. Comm. 1997. V. 230, P. 285-288), cDNA synthesis method matrix change (Chenchik et al., (1998) Gene Cloning and Analysis by RT-PCR, ed. by Ρ Siebert and J Larrick, BioTechniques Books, Natick, MA, 305-319; Zhu et al. Biotechniques. 2001.30, 892-897) and others.

[075] In some applications, the DNA sample is a sample of tumor or fetal DNA or cDNA present in a biological fluid of a human or animal as part of circulating tumor or fetal cells, plasma microvesicles, complexes with proteins, carbohydrates and lipids, or in free form. It is known that DNA of tumor origin is contained in biological fluids in very small amounts even with metastatic tumors (Diehl, F, et al. Gastroenterology. 2008 Aug; 135 (2): 489-98). To enrich a sample of such DNA, the biological material may be pretreated. For example, a method for separating blood plasma from blood cells can be used for subsequent analysis of low-represented mutations of tumor or fetal origin (as described, for example, in: Lo, YMD, et al. Clin Chem. 1999 Oct; 45 (10): 1747-51 ) In another application, DNA or RNA can be isolated from blood serum (as described, for example, Anker, P., et al. Cancer Metastasis Rev. 1999; 18 (1): 65-73). In some applications, fractionation of the cell sediment of the biological fluid can also be carried out in order to separate the fraction of tumor or fetal cells by separation using antibodies on flow cytometers (as described, for example, in: Min, A. et al. Clin Exp Metastasis. 2009; 26 (7): 759-67) or using magnetic particles (as described, for example, in: Bilkenroth U., et al. Int J Cancer. 2001 May 15; 92 (4): 577-82), filtering through a membrane ( as described, for example, in: Takano T., et al. Head Neck. 2008 Aug; 30 (8): 983-90), density gradient centrifugation (as described, for example, in: Rosenberg, R. et a l. Cytometry. 2002 Dec 1; 49 (4): 150-8) or dielectrophoresis (as described, for example, in: Gascoyne, P. et al. Electrophoresis. 2009 Apr; 30 (8): 1388-98). The fraction of microvesicles (exosomes, “virtosomes” and others) potentially containing nucleic acids can be separated from biological fluids by ultracentrifugation, ultrafiltration or separation using antibodies (as described, for example, in Taylor, DD, et al. Methods Mol Biol. 2011; 728: 235-46; Rood, IM, et al. Kidney Int. 2010 Oct; 78 (8): 810-6; Clayton, Α., Et al. J Immunol Methods. 2001 Jan 1; 247 (1-2): 163-74; Gahan, P. B., Stroun, M. Cell Biochem Funct. 2010 Oct; 28 (7): 529-38). In some applications, nucleic acids bound to the surface of blood cells or other body fluids (as described, for example, Laktionov, PP, et al. Ann Ν Acad Sci) can be isolated by incubating cells in a buffer containing trypsin and EDTA salts. 2004 Jun; 1022: 221-7; Mal'shakova, VS, et al. Ann Ν Y Acad Sci. 2008 Aug; 1137: 47-50). In some applications, tissue preparations can be enriched with target cells using laser microdissection (as described, for example, in: Cha, S., et al. Mol Cell Proteomics. 2010 Nov; 9 (11): 2529-44; Weier, JF , et al. Cytogenet Genome Res. 2006; 114 (3-4): 302-11).

[076] In some applications, the enrichment of a sample of tumor or fetal DNA can be carried out after the extraction of DNA from biological fluids. For example, to increase the concentration of fetal DNA in DNA samples isolated from the blood plasma of pregnant women, the sample can be treated with formaldehyde, as proposed by Dhallan et al. (Dhallan, P., et al. JAMA. 2004. Vol. 291. No. 9. P . 1114-1119). Also, to increase the concentration of fetal or tumor DNA in the sample, it can be used that such DNA is represented almost exclusively by low molecular weight fractions (Li, Y., et al. Methods Mol Biol. 2008; 444: 239-51; Hung, Ε.С, et al. Clin Chem. 2009 Apr; 55 (4): 715-22; Hahn, T., et al. Clin Chem. 2009 Dec; 55 (12): 2144-52) and this fraction can be separated from other fractions on agarose electrophoresis or on a chromatographic column.

[077] In preferred applications, the DNA sample for analysis contains the target nucleic acid (ie, mutant DNA, reference DNA, or a mixture thereof). In some applications, the nucleic acid sample also contains non-target nucleic acids that are not similar to the target nucleic acids. As used here, the term "non-target" refers to a nucleic acid that does not contain the test nucleotide and is not complementary to the target nucleic acid.

[078] In preferred applications, the concentration of nucleic acids in the sample is at least 10 pkg, usually at least 500 pkg. The proportion of the target nucleic acid in the sample can range from 0.0000001% to 100% and most often does not exceed 50%, mainly does not exceed 10%, as a rule, does not exceed 1-2%.

[079] In some applications, the DNA sample contains an excess of the reference target sequence compared to the mutant. Reference (non-mutant) DNA is in more than 100-fold excess, for example, more than 300-fold excess, usually more than 500-fold excess, mainly more than 1000-fold excess, for example, a 10,000-fold excess. In some applications, the reference DNA is in more than 1,000-fold excess, but not more than in 100,000-fold excess.

[080] In some embodiments, the DNA sample is fragmented to obtain DNA fragments with an average length of from 50 to 1000 nucleotides, more often from 100 to 500 nucleotides, for example 150-300 nucleotides.

[081] In some embodiments, exogenous double-stranded DNA from another organism or artificially created is added to a DNA sample isolated from biological material. Amplification of fragments of this DNA can be further used as quality control of amplification and sequencing. For example, invertebrate DNA or an artificially generated sequence can be added to a DNA sample isolated from a human. Examples of such sequences are shown in SEQ ID NOs: 64, 65.

[082] Molecular barcoding of a pool of target DNA molecules

[083] A DNA sample is used as a matrix for barcoding a pool of target molecules. As used here, the term "barcoding" means the introduction of a barcode sequence into a DNA sequence. As used here, the term "barcode" means a nucleotide sequence that allows you to distinguish between the amplification product of one molecule and the amplification product of other identical or essentially similar molecules. As used here, the term "barcode" does not refer to index sequences that serve to distinguish molecules from two different DNA samples.

[084] For the needs of the present invention, the process of barcoding consists in introducing a random sequence of nucleotides (random primer) in the target DNA molecule. The length of the random sequence can vary and is usually 9-20 nucleotides, usually 10-15 nucleotides, for example 12, 13 or 14 nucleotides. As used here, the term "barcoding primer" means a mixture of oligonucleotides containing a random sequence - barcode. The structure of the barcoding primer is shown schematically in Fig. 1. At the 3'-end, the primer carries a gene-specific sequence complementary to the target DNA sequence, followed by a random sequence (barcode). In preferred embodiments, behind the barcode (at the 5'-end) is an adapter sequence.

[085] Examples of barcoding primers for the target sequences of SEQ ID NOS: 1-62 are shown in SEQ ID Nos: 66-127. These primers contain gene-specific sequences adjacent to the reference sequences shown in SEQ ID NOS: 1-62. The correspondence of the primers to the target sequences is shown in Table 1.

[086] It is obvious to any person skilled in the art that the length of the barcode can be reduced or increased by 1-4 nucleotides without a significant impact on the efficiency of barcoding. It is also apparent that the adapter sequence may be replaced. Variants of adapter sequences are well known and shown, for example, in the UniVec database provided by the National Center for Biotechnology Information, in instructions for next-generation sequencing instruments and new-generation sequencing kits.

[087] The introduction of barcode into the pool of target molecules is carried out during the polymerase chain reaction (PCR) in the presence of a barcoding primer at a final concentration of 0.03-0.3 μM, usually 0.05-0.2 μM. Preferred is the use of DNA polymerase or a mixture of DNA polymerases, characterized by increased accuracy of synthesis and specificity of the seed of synthesis - for example, polymerase, which contains additional amino acid substitutions that enhance the accuracy of synthesis (for example, described in: Lovmar, L., Syvaenen, A.C. Methods Mol Med. 2005; 114: 79-92), such as, for example, Tersus (ZAO Evrogen, Russia), Phusion (New England Biolabs, USA), Kapa Hi-Fi (Kapa Biosystems, South Africa), TruSeq DNA Polymerase (Illumina, USA), KOD DNA Polymerase (TaKaRa Bio, Japan), or the like.

[088] Barcoding can be performed during standard PCR as described by Kinde et al. (Proc Nat Acad Sci USi. 2011; 23: 9530-35). In this case, the PCR reaction mixture also contains a counter gene-specific primer for each barcoding primer. The terms “forward primer” and “counter primer” are used here to indicate the relative position of two primers on a DNA molecule, but without regard to their position relative to the DNA coding chain. Examples of counter gene-specific primers for amplification of reference sequences of SEQ ID NOS: 1-62 are shown in SEQ ID NOs: 131-192. The gene-specific sequences of these primers are distant from the reference sequences of SEQ ID NOs: 1-62 per 10-25 nucleotides. The correspondence of reference sequences and gene-specific primers is shown in table 1.

[089] As an alternative, Applicants have developed an original linear PCR barcoding procedure in the presence of only barcoding primers (Fig. 2). During such PCR, the matrix is only the original DNA, which allows you to increase the number of amplification cycles to 15, more often to 5-7. Thus, during barcoding, each molecule of the initial sample corresponds to at least 5 copies, each of which carries its own barcode.

[090] This makes it possible to further identify DNA polymerase errors occurring at this stage: with a sufficient number of linear amplification cycles, a mutation present in at least one molecule of the initial sample should be present in several products of this reaction containing different barcodes (expected number of these products is calculated taking into account the effectiveness of specific reactions and the number of amplification cycles). If the number of these products is more than 3, then the likelihood of the appearance of an equally frequent artifact mutation is extremely small, and DNA polymerase errors at the initial stage can be detected using "filtering by quantity."

[091] Each amplification cycle includes denaturation at a temperature of 94-96 ° C, annealing of primers at a temperature of 50-65 ° C, and elongation at a temperature of 72 ° C. The choice of amplification program is well known to those skilled in the art. The annealing temperature is chosen in accordance with calculations in the M-Fold (Zuker, M. Nucleic Acids Res. 2003 Jul 1; 31 (13): 3406-15), MeltCalc (Schuetz, E., von Ahsen, N. Biotechniques. 1999 programs Dec; 27 (6): 1218-22, 1224) or the like and are tested experimentally on control DNA samples.

[092] Depending on the number of target sequences to be analyzed in the sample, barcoding can be performed in one tube during multiplex linear PCR or in several tubes during multiplex linear PCR, where each tube contains its own set of barcoding primers.

[093] In some embodiments of linear multiplex PCR, various barcoding primers are present in equimolar amounts in the reaction mixture. In some embodiments, the concentrations of some primers are increased or decreased (for example, 1.5-3 times) relative to others in such a way as to maintain a close to the original ratio of the concentrations of target molecules at the end of sample preparation. For each set of primers, experimental selection of the optimal concentration ratio is necessary.

[094] In some embodiments, each mixture of barcoding primers also contains primers for barcoding the "internal control" - a gene sequence that is not mutant for a given sample. For example, in the case of using human tumor DNA, the IDH1 gene fragment, the sequence of which is shown in SEQ ID No: 63, can be used as the internal control matrix. The sequence of the barcoding primer for this fragment is shown in SEQ ID No: 128.

[095] In some embodiments, each mixture of barcoding primers also contains primers for barcoding the exogenously added DNA. Examples of primers for barcoding fragments of SEQ ID Nos: 64, 65 are shown in SEQ ID Nos: 129, 130.

[096] Purification of the product of the barcoding reaction from the residues of the barcoding primer

[097] Purification of the barcoding product from the remainder of the primer is designed to exclude the participation of the primer containing barcode in further amplification. For purification, any known method for purifying DNA from short oligonucleotides, including gel filtration and enzymatic methods, or a combination thereof, can be used. In particular, commercially available reagent kits for purifying DNA from reaction mixtures, such as the Cleanup Standard kit (Eurogen, Russia), DNA Clean & Concentrator-5 (Zymo Research, USA) and the like, can be used.

[098] In some applications, exonuclease I (Exo-I), which destroys single-stranded DNA in the 3'-5 'direction, releasing deoxyribonucleoside-5-monophosphates can be treated to destroy residues of the barcoding primer. Exo-I is active in the PCR reaction buffer and can be added to the reaction mixture immediately after completion of the barcoding procedure. Typically, Exo-1 is used in a final concentration of 1 to 3 units. in μl, for example, 2.4 units in μl.

[099] Exo-I treatment is carried out at 37 ° C. Processing time can vary from 5 minutes to 2 hours, more often ranging from 7 minutes to 1 hour, for example 10-15 minutes, which is sufficient for hydrolysis of the residues of the barcoding primer, but does not affect the target sequences of the barcoded DNA molecules (Fig. 2).

[0100] Inactivation of Exo-I is carried out by heating at 68-70 ° C for 10-30 minutes.

[0101] In some embodiments, the reaction product after Exo-I treatment may be reprecipitated. In some embodiments, the reaction product after Exo-I treatment can be purified using commercially available DNA purification reagent kits from reaction mixtures or any other known method for DNA purification.

[0102] Amplification of the pool of target molecules in multiplex PCR

[0103] Amplification of the pool of barcoded target molecules is carried out in standard PCR using a universal primer containing at the 3 'end a sequence essentially similar to the adapter sequence of the barcoding primer and a gene-specific primer that is counter to the gene-specific sequence of the barcoding primer (Fig. 2) . For example, the TS-Uni-short universal primer, the sequence of which is shown in SEQ ID No: 196 and counter genespecific primers selected from SEQ ID NOs: 131, can be used to amplify barcoded molecules using primers selected from SEQ ID NOs: 66-130 -195.

[0104] The number of PCR tubes is equal to the number of tubes used for barcoding. Each contains its own set of gene-specific primers (hereinafter referred to as REV primers), which are counter to barcoding primers used in the previous stage (table 1).

[0105] The volume of the reaction mixture for PCR is typically 10-100 μl, usually 15-50 μl, for example, 20 μl or 25 μl. As a matrix in PCR, a purified barcoding product is used.

[0106] The final concentration of universal primer in the reaction mixture is usually 0.2-0.5 μM, for example 0.3 μM. The final concentration of each gene-specific primer is 0.01-0.3 μM, usually 0.03-0.2 μM.

[0107] In some embodiments, various gene-specific multiplex PCR primers are present in equimolar amounts in the reaction mixture. In some embodiments, the concentration of some primers is increased or decreased (for example, 0.5-3 times) relative to others in such a way as to maintain close to the original ratio of the concentrations of target molecules in the amplified sample. For each set of gene-specific primers, experimental selection of the optimal concentration ratio is necessary.

[0108] In some embodiments, each PCR mixture also contains gene-specific primers for amplification of the "internal control" and / or exogenously added DNA. For example, for the reference sequences shown in SEQ ID Nos: 63-65, gene specific REV primers shown in SEQ ID Nos: 193-195 can be used.

[0109] Amplification is carried out according to the following program: 25-90 PCR cycles in 1-30 sec mode — denaturation at a temperature of 94-96 ° C, 40 sec — 1 minute — annealing of primers at a temperature of 50-65 ° C, elongation for 1 minutes at 72 ° C. The annealing temperature is selected in accordance with the calculations in the M-Fold program (Zuker, M. Nucleic Acids Res. 2003 Jul 1; 31 (13): 3406-15), MeltCalc (Schuetz, E., von Ahsen, N. Biotechniques. 1999 Dec; 27 (6): 1218-22, 1224) or the like and are tested experimentally on control DNA samples.

[0110] the Introduction of sequence adapters in the product of multiplex PCR and sequencing of the specified product

[0111] Sequencing of the amplification product can be performed by any known sequencing method, including Sanger sequencing and new generation high-performance sequencing.

[0112] For the method of the present invention, sequence adapters for NGS can be introduced into the amplification product in various ways known to those skilled in the art, including ligation or various PCR variants with primers containing sequences of sequence adapters at the 3'-end. Various methods of introducing sequence adapters during one or more PCR are illustrated in Fig. 3.

[0113] In some embodiments, the introduction of sequencing adapters is carried out using PCR with primers containing sequences of sequenced adapters at the "hanging" 5'-ends. The 3'-terminal sequences of the primers contain annealing sites for the target molecules, for example, for gene-specific sequences within the amplified regions of the target molecules (in-depth PCR) and for the adapter sequence of the barcoding primer.

[0114] As used here, the term "hanging end" or "hanging tail" in relation to the primer refers to the 5'-terminal nucleotide sequence in the primer, non-complementary to the target molecules.

[0115] In some embodiments, sequencing adapters are introduced using two consecutive PCRs, where primers containing 3'-terminal annealing sites to target molecules and hanging ends containing part of the sequence of the sequencing adapter or any other sequence are used during the first PCR. The second PCR uses primers containing the 3'-terminal annealing site at the hanging ends of the previous PCR primers and sequences of sequence adapters at the 5'-end.

[0116] In some embodiments, sequences of sequence adapters are introduced as hanging ends in the sequence of primers used in the multiplex PCR step. In this case, the introduction of sequence sequences is carried out immediately at the stage of multiplex PCR.

[0117] In some embodiments, Step-Out PCR (Matz et al. Nucleic Acids Res. 1999; 27 (6): 1558-1560) is used to introduce sequencing adapters. For amplification, two primer mixtures are used, each of which consists of (1) an “internal” primer carrying an annealing site at the 3 'end to the target sequences (3'-end specific sequence) and a drooping tail at the 5'-end and (2) An “external” primer containing at the 3 ′ end a sequence identical to the sequence of the hanging tail of the “internal” primer. Concentrations of primers in the mixtures are selected so that the “internal” primer is consumed in the first PCR cycles, during which molecules are created that have sites for annealing the “external” primer. In subsequent PCR cycles, amplification of the pool of target molecules continues from the “external” primer. For example, the final concentration of each internal primer is usually 0.05-0.3 μM, for example 0.1 or 0.2 μM, and the concentration of the external primer is 10-15 times higher.

[0118] An index sequence may also be included in the primers. For amplification of various samples, primers are used that carry different index sequences, which subsequently makes it possible to distinguish molecular sequencing data from different samples. The length of the index sequence is usually 3-10 random nucleotides, for example, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides. Examples of such index sequences are available on the websites of reagent kit manufacturers for high throughput sequencing.

Identification of mutations in target DNA by sequencing data

[0119] The total error rate generated during multi-stage sample preparation and sequencing is at least 1% (Wei, Ζ., Et al. Nucleic Acids Res. 2011 Oct; 39 (19): e132; Klein, JD, et al. PLoS One. 2011; 6 (8): e23455). Thus, for reliable identification of rare mutations (for example, onc mutations), analysis of sequencing data requires screening of false-positive signals - replacing nucleotides introduced into the sequence as a result of incorrect operation of DNA polymerase at the PCR stages and incorrect readings during sequencing.

[0120] In preferred embodiments, screening of false-positive signals includes barcode sequence analysis. The presence of the same barcode in molecules means that they are descendants of the only barcoded DNA matrix molecule. The descendants of one molecule must have the same mutation profile. The presence of various variants of the nucleotide sequence among the descendants of one molecule indicates that either all of these variants, or all of these variants, except for one, represent an unreliable (more often false positive) signal.

[0121] In some embodiments, screening of false-positive signals also includes "filtering by quantity", which allows excluding from consideration descendants of a barcoded molecule containing a substitution resulting from malfunctioning DNA polymerase at the barcoding stage.

[0122] In some embodiments, false positive screening also includes PCR error screening using the MIGEC strategy described by Shugay et al. (Shugay et al. Nat Methods. 2014; 11 (6): 653-5).

Primers for the analysis of target regions in key human oncogenes and human suppressor genes

[0123] Also provided are primer sets for implementing the method of the present invention when analyzing mutations in the gene sequences shown in SEQ ID No: 1 to SEQ ID No: 62. According to literature data, mutation analysis in these regions is in demand for identifying and monitoring a number of oncological diseases, and also has high prognostic and predictive value in the selection of strategies and tactics for managing patients with similar diseases.

[0124] The barcoding and amplification primers are divided into groups (Table 2), each of which can be used to simultaneously barcode and amplify target fragments in a single tube. For any specialist in this field it is obvious that some primers can be excluded from the group if there is no need to analyze any of the listed fragments. Such a decrease in the number of primers in the mixture does not affect the efficiency of barcoding and amplification of the remaining fragments.

[0125] In preferred embodiments, primers for multiplex PCR1 are used in conjunction with a counter primer whose sequence is shown in SEQ ID No: 196.

[0126] Primers are also provided for preparing mixtures for introducing sequence adapters using PCR to the outside. Mixture 1 includes an internal primer having an annealing site for the adapter sequence of the barcoding primer, common to all barcoding primers, and a “hanging tail”, and an external primer containing the sequence of the sequencing adapter and a sequence identical to the “hanging tail” of the internal primer. Examples of such Illumina sequencing primers are shown in SEQ ID Nos: 197, 198.

[0127] Mixture 2 includes a set of internal primers containing a 5'-terminal "hanging tail" and a 3'-terminal gene-specific sequence, recessed relative to the gene-specific primers for multiplex PCR and adjacent to the reference sequence of SEQ ID NOs: 1-62 (BRIDGE primers ) Mixture 2 also includes an external primer containing the hanging tail sequence of the internal primer at the 3'-end and the sequence of the sequence adapter at the 5'-end. After the sequence of the sequence adapter (from 5 'to 3'), the specified external primer may also contain an index sequence. Examples of internal primers are given in SEQ ID NOs: 199-260, examples of external primer in SEQ ID NO: 264, 265.

[0128] It is obvious to any person skilled in the art that the index sequence can be replaced in such a primer with any other nucleotide sequence of the same or close length. Also, the index sequence can be transferred to the external primer of mixture 1. Also, sequences of sequence adapters can be replaced in the external primers in accordance with the requirements of sequencer manufacturers for high-performance sequencing. The following examples show sequencing adapters for sequencing on Illumina sequencers.

[0129] External and internal primers can also be used to introduce sequencing adapters during two rounds of PCR as shown in Fig. 3. In this case, the internal primers are used for the first round of PCR (at a concentration of 0.05-0.3 μM), and the external ones for the second round of PCR.

[0130] In some embodiments, the primers in each group are mixed in equimolar amounts. The final concentration of each primer is 0.7-0.15 μM, for example 0.1 μM. In some embodiments, the concentration of some primers is increased by 1.5-5 times compared with the rest. In some embodiments, the concentration of some primers is reduced by 1.5-5 times compared with the rest. The concentrations of the external primers in the PCR mixtures with the exit to the outside exceed the concentrations of the internal primers by at least 5 times, more often 10 times, sometimes 15 times.

[0131] For example, mixtures of barcoding primers A, B, C, and D are provided (Table 2), where the concentration of barcoding primers is at least doubled for the following reference fragments: exon 4 of the HER4 gene, exon 12 fragment 2 of the HER4 gene, exon 14 HER4 gene, exon 8 of the HER3 gene, exon 21 of the HER3 gene, exon 23 of the HER3 gene, exon 10 of the PIK3CA gene, exon 4 of the KRAS gene, exon 2 of the AKT1 gene, exon 4 of the MAP2K2 gene, exon 5 of the GNAQ gene, exon 2 of the RAC1 gene. Mixtures of REV primers for multiplex amplification of barcoded molecules are also provided, where the concentration of gene-specific primers for amplification of the following reference fragments is at least doubled: exon 4 of the HER4 gene, exon 12 fragment 2 of the HER4 gene, exon 14 of the HER4 gene, exon 8 of the HER3 gene, exon 21 HER3 genes, exon 20 fragment 1 of the EGFR gene, exon 8 fragment 2 of the HER4 gene, exon 5 fragment 2 of the TP53 gene, exon 23 of the HER3 gene, exon 10 of the PIK3CA gene, exon 4 of the KRAS gene, and the concentration was reduced by at least a factor of 1.5 for amplification of the following reference fragments c: exon 7 of TP53 gene, exon 18 of EGFR gene, exon 21 of EGFR gene, exon 20 fragment 1 of HER2 gene, exon 21 fragment 1 of HER2 gene, exon 9 of HER3 gene, exon 3 of HER3 gene, exon 11 of KIT gene, exon 15 of BRAF gene , exon 2 of the NRAS gene, exon 20 of the PIK3CA gene, exon 3 KRAS, exon 2 and exon 3 of the HRAS gene, exon 3 of the NRAS gene, exon 12 of the EGFR gene, exon 7 of the RAF1 gene, exon 2 of the MAP2K1 gene, exon 6 of the MAP2K2 gene, exon 5 GNAQ gene, exon 7 of the MAPK1 gene.

[0132] In preferred embodiments, the primer mixtures also contain primers for barcoding and amplifying an “internal control”, for example, exon 4 of the IDH1 gene (SEQ ID NOs: 63). An analysis of the amplification and sequencing data of this gene can serve as a control of the quality of the analysis. Examples of such primers are shown in SEQ ID NOs: 128, 193, 261.

[0133] In preferred embodiments, the primer mixtures also comprise primers for barcoding and amplifying exogenously added sequences. Examples of such primers are shown in SEQ ID NOs: 129, 130, 194, 195, 262, 263.

[0134] In some embodiments, the primer mixture contains additional primers for amplification of other additional sequences, selected in such a way that they do not interfere with the amplification of the above reference sequences.

[0135] A mixture of primers can be used to implement the method of the present invention, as described in the "Examples" section below.

[0136] Mixtures of barcoding primer primers can be used for standard multiplex PCR DNA fragments containing reference regions of SEQ ID NOs: 1-62 together with their corresponding REV primers. The products of such PCR can be used as a matrix for in-depth multiplex PCR with BRIDGE primers and TS-Uni-short primers or barcoding primers.

Reagent kits

[0137] Reagent kits for implementing the method of the present invention are also provided. In preferred embodiments, said kits include mixtures of barcode primers and multiplex amplification. In some embodiments, the kits also include primers for introducing sequencing adapters by PCR to the outside. In some embodiments, the kits also include PCR reagents. In some embodiments, the kits also include reagents for purifying DNA from the reaction mixtures and exonuclease I.

[0138] In preferred embodiments, the kits also include instructions for use on paper or electronic media containing a protocol for sample preparation and analysis of sequencing data.

Application methods

[0139] Possible applications of the invention include all areas of biology and medicine in which the identification of DNA or RNA molecules with mutations and polymorphisms may be relevant, especially those present in samples containing an excess of non-mutated DNA or RNA molecules.

[0140] Such applications include, but are not limited to, the following areas:

[0141] Detection of somatic mutations, first of all, non-invasive screening and monitoring in oncology, including early diagnosis of malignant tumors and their relapses, assessment of the radical nature of the treatment, diagnosis of micrometastases and "sleeping metastases" and evaluation of the drug sensitivity of the tumor without biopsy;

[0142] Non-invasive prenatal diagnosis;

[0143] DNA diagnosis of de novo mutations and diseases caused by chimerism;

[0144] Monitoring recipients of transplanted organs for timely detection of transplant rejection;

[0145] Identification of acquired drug resistance of infectious agents and malignant tumors, including directly during therapy;

[0146] DNA diagnosis of mitochondrial diseases (in most cases, such diseases are caused by changes in the mitochondrial genome that are not present in all mitochondria - the so-called heteroplasmy);

[0147] Analysis of the level of spontaneous mutagenesis for a wide range of tasks in biology and medicine;

[0148] Analysis of genetic polymorphism on the pulled samples (in particular, in population, evolutionary and environmental genetics).

[0149] The following examples are provided as illustrative, but not limiting.

EXAMPLES

[0150] Example 1

[0151] Comparison of the effectiveness of different methods of barcoding target molecules

[0152] Barcoding of the molecules was performed using standard PCR and linear PCR. To prepare the reaction mixtures, Tersus PCR kit reagents were used (Eurogen, Russia), according to the manufacturer's instructions. Two reaction mixtures were prepared (each volume was 25 μl): the mixture for barcoding in PCR with two primers contained 2 μM primer SEQ ID No: 100 and 2 μM primer SEQ ID No: 165, the mixture for barcoding in PCR with one primer contained 2 μM primer SEQ ID No: 100. 10 ng of human genomic DNA was added to each reaction mixture (Clontech, USA). The mixture was amplified in the following mode: preliminary denaturation at 95 ° C for 5 min; 2 cycles: 95 ° C - 15 sec; 58 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes At the end of the reaction, both mixtures were incubated with 3 μl of exonuclease I (New England Biolabs) for 1 hour at 37 ° C, heated for 15 min at 70 ° C to inactivate the enzyme, and amplified in the presence of TS-Uni-short primers and SEQ ID No: 165 ( 2 μM per reaction) in PCR in the following mode: preliminary denaturation at 95 ° C for 5 min; 28 cycles: 95 ° C - 15 sec; 62 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes PCR products were diluted 40 times and amplified (1 μl of the diluted product per 50 μl of the PCR reaction mixture) in the presence of TS-Uni-short and SEQ ID NO: 233 primers using the Tersus PCR kit (Eurogen, Russia) according to the attached instructions . The following amplification mode was used: preliminary denaturation at 95 ° C for 5 min; 12 cycles: 95 ° C - 15 sec; 62 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes PCR products were allowed for agarose gel electrophoresis in TAE buffer stained with ethidium bromide (Fig. 4). It can be seen that in the case of barcoding using linear PCR, target amplification is more efficient.

[0153] Example 2

[0154] Analysis of mutations in the BRAF gene

[0155] To create the experimental samples, a commercial human genomic DNA sample was used from healthy male donors (Human Male Control Genomic DNA, Promega, Cat No. G1471) - hereinafter referred to as “BRAF-wt sample”. To obtain BRAF mutant DNA, the genomic DNA of the SK-Mel-1 cell line containing a heterozygous mutation c. 1799 T → A (p. Val600Glu) in the BRAF gene.

[0156] To isolate DNA, the SK-Mel-1 cell line was incubated with proteinase K (Amresco, Cat No. O706) in standard buffer (100 mM Tris-HCl; 50 mM K3EDTA; 0.5% SDS; pH = 8.0 ) Aliquots of 1 million cells were incubated in 1.2 ml of buffer for 3 days at 56 ° C. DNA was isolated from the lysate using the DNA Clean & Concentrator-5 reagent kit (Zymo Research, Cat. No. D4013) according to the manufacturer's instructions, 300 μl of the lysate was used for one isolation.

[0157] DNA samples were subjected to ultrasonic fragmentation, the fragmentation results were monitored by agarose gel electrophoresis stained with ethidium bromide. Fragmentation was carried out until dominant fragments from 100 to 150 bp in length appeared. DNA concentration was measured using an XY-Detect reagent kit (Synthol, Russia, cat. No. HG-402) and a panel of DNA calibrators (at a concentration of 50, 10 and 1 ng / μl; Isogen Laboratory, Russia, cat. No. R0010) in according to manufacturers instructions.

[0158] To obtain amplified experimental samples, BRAF-wt DNA was mixed with the DNA of the SK-Mel-1 cell line in a ratio of 100 to 1 (sample BRAF-mut100) and 1000 to 1 (sample BRAF-mut1000). As a control, a BRAF-wt DNA sample was used. The mutational status of the BRAF gene in BRAF-mut100, BRAF-mut1000, and BRAF-wt DNA samples was confirmed by standard and mutational-specific real-time PCR using the Insider B-Raf reagent kit (Eurogen, Russia) followed by Sanger sequencing. Mutant DNA was shown to be in 30 and 300 fold excess in samples BRAF-mut100, BRAF-mut1000, respectively.

[0159] Samples of BRAF-wt DNA, BRAF-mut100 and BRAF-mut1000 were used as a matrix for barcoding the target exon 15 fragment of the BRAF gene. Barcoding using linear PCR, purification and subsequent amplification of the target fragment was carried out as described in Example 1. The only difference was that 3 cycles of linear PCR were used for barcoding. The PCR product was diluted 40 times and 1 μl of the diluted product was added to 50 μl of the PCR reaction mixture, which was carried out in the presence of primers SEQ ID NOs: 197 and 233 in the preliminary denaturation mode at 95 ° C for 5 min; 17 cycles: 95 ° C - 15 sec; 62 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes PCR products were diluted 40 times and 1 μl of the diluted product was added to 50 μl of the PCR reaction mixture, which was carried out in the presence of primers SEQ ID NOs: 198 and one of 264, 266, 267 (primers with different index sequences, different for each sample) in the mode of preliminary denaturation at 95 ° C for 5 min; 17 cycles: 95 ° C - 15 sec; 58 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes The amplification efficiency was monitored using agarose gel electrophoresis in TAE buffer (40 mM Tris acetate, pH 7.8-8.0; 1 mM EDTA) stained with ethidium bromide. The results of gel electrophoresis are summarized in Fig. 4. PCR products of different samples were purified using a PCR product purification kit (Qiagen, Germany), the product concentration was measured on a Qubit 2.0 fluorimeter (Life Technologies, USA) using a Quant-IT dsDNA BR Assay Kit (Life Technologies, USA, Cat. ID Q32853) according to the manufacturer's instructions and 700 ng of DNA of each product were combined and sequenced on a MiSeq v. 2 (Illumina) using the MiSeq 2 X 150 Cycle Sequencing Kit v. 2 (Illumina, Cat. No. MS-102-2002) in accordance with the manufacturer's instructions. Paired (double-sided) readings of 150 bases were obtained.

[0160] The results of massive sequencing were automatically sorted according to samples according to the labels of the samples and used to analyze mutations in exon 15 of the BRAF gene. At the first stage of data analysis, data quality was evaluated according to a standard protocol using fastqc software with further filtering using flexbar software. Further, sequencing results (individual readings) were mapped onto the reference sequence of the 15th exon of the BRAF gene (SEQ ID NO: 1). In the first case, readings were mapped using the Bowtie2 program using standard parameters. The data obtained at this stage corresponded to the standardly obtained result of bioinformation analysis. In the second case, first readings were filtered out that did not overlap the reference sequence or did not contain a full barcode. The resulting data pool was grouped into “families” within which all molecules had the same barcode. Families containing one member were discarded as unsuitable for statistical analysis. As a result of data processing, taking into account all the requirements, ~ 20% of reads were dropped. After the formation of “families” with identical barcodes for each of them, with the help of the Bowtie2 program, consensus was collected with the dominant variant in each position (the share of the dominant variant is at least 95%). If the dominant variant was not found in the consensus sequence, the symbol “X” was introduced. Finally, the obtained consensus sequences were mapped onto the reference sequence of the 15th exon of the BRAF gene using the Bowtie2 program using standard parameters.

[0161] The alignment results before and after processing were transformed into a table containing data on how many times each variant occurred in each position of the sequence. To calculate the approximate number of molecules of the target DNA fragment in PCR products and in the original samples, we used the obtained data on the number of reads and families with different barcodes, respectively, adjusted for the total volume of the reaction mixture: for samples used for sequencing: the total volume of the reaction mixture was 60 times the volume used for sequencing (table 3).

[0162] To assess the selectivity of the method, the presence of a “signal” in the data for samples with mutations (readings and families corresponding to the mutation p. 1799 T → A) was checked, and if this signal was present, its level was compared with the “noise” level from PCR and sequencing errors. The noise level K was understood as the ratio of the number of readings with incorrect options to the sum of the number of readings with incorrect options and the number of readings with the correct option (readings with the symbol "N" in both cases were not taken into account either in the numerator or denominator; readings with the symbol "X" in the case of equalization with preliminary clustering by barcodes, they were taken into account as part of the noise, i.e., both in the numerator and denominator). The obtained series of values of the parameter K (a total of 12 series) were analyzed using basic statistical methods. For each series, the sample mean and standard deviation of the sample were calculated. The correspondence of the distribution of values in each row to the normal distribution was checked using the Kolmogorov-Smirnov criterion. Since, according to this criterion, the distribution corresponded to the normal one, we compared the average values in pairs of rows for one sample using the Student t-test (two-sided, two-sample with unequal deviation). Statistical operations were performed using the Microsoft Excel program and the AtteStat add-in (StatSoft, Russia). The results of the statistical analysis are summarized in Table 4. Alignment with pre-clustering by barcodes reduced the noise level by about 10 times, and this difference is characterized by a high level of statistical significance (p-value). In parallel with a decrease in the noise level, the signal level increases, and, accordingly, the selectivity of the analysis increases. If in a standard analysis the signal level exceeds the average noise level by only 3.5 or 4.6 times, then after clustering by barcodes the signal exceeds the average noise level by 31 times or more. Thus, although the signal corresponding to the presence of a mutation c. 1799 T → A, was found in the data of all samples, using standard equalization, it is possible to reliably distinguish the signal from noise with standard alignment only in the BRAF-mut100 sample. In other words, with standard alignment, a selectivity level of 1:30, but not 1: 300, is achieved.

[0163] Example 3

[0164] Verification of the specificity of the primers

[0165] Oligonucleotide primers (Table 1) were synthesized on an Applied Biosystems ABI 3900 synthesizer. Oligonucleotides were desalted and purified on a SDS page according to a standard protocol. To prepare mixtures of oligonucleotides, lyophilized primers obtained after synthesis were dissolved with sterile deionized water for molecular biological work to a final concentration of 10 μM. Oligonucleotides for each group (Table 2) were mixed in equimolar amounts and used for test PCR. In each case, a 25 μl reaction mixture containing a mixture of barcoding primers (groups A, B, C or D) in a final concentration of 0.2 μM each, 1X Tersus polymerase buffer (Eurogen, Russia), 10 ng of human genomic DNA (Human Male Control Genomic DNA, Promega, Cat No. G1471), 200 μM of each nucleoside triphosphate and Tersus polymerase were subjected to linear PCR in the mode: preliminary denaturation at 95 ° C for 5 min; 5 cycles: 95 ° C - 15 sec; 58 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes Next, 1.5 μl of endonuclease I (New England BioLabs) was added to the reaction mixture and incubated for 45 min at 37 ° C. The endonuclease was inactivated for 10 min by heating at 68 ° C and the tube was transferred to ice. After 2 min, reaction products were purified using agarose gel DNA extraction kits and Cleanup Mini reaction mixtures (Eurogen, Russia), 11.5 μl of sterile deionized water was eluted for molecular biological work. 10 μl of the obtained product was used for amplification in the presence of a mixture of REV primers for multiplex amplification (groups A, B, C or D, respectively) and TS-Uni-short primer (final concentration of primers 0.2 μM). For PCR, a Tersus PCR kit reagent kit (Eurogen, Russia) was used according to the attached instructions. Amplification was carried out in the following mode: preliminary denaturation at 95 ° C for 1 min; 29 cycles: 95 ° C - 15 sec; 62 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes The PCR product (final dilution in the new reaction mixture 1: 1000) was subjected to nested PCR in the presence of a mixture of genespecific internal PCR primers with outward passage (mixture 2) and TruSeq Universal long primer (SEQ ID No: 197) at a final concentration of 0.2 μM in the mode: preliminary denaturation at 95 ° C for 1 min; 17 cycles of PCR: 95 ° C - 15 sec; 58 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes

[0166] The obtained PCR product was diluted (final concentration in the new reaction mixture 1: 1000) and the specificity of amplification was analyzed using individual primer pairs (barcoding primer + gene-specific BRIDGE primer) for each reference fragment (table 1). For PCR, the Encyclo PCR kit (Eurogen, Russia) was used according to the attached manufacturer's instructions. Amplification products were analyzed by agarose gel electrophoresis with TAE buffer and ethidium bromide staining. The PCR test products were cloned into a pAL-TA vector, and in each case, the inserts of three selected clones were sequenced to confirm that the structure of the cloned fragment matches the expected nucleotide sequence. Sequencing was performed on an ABI3730xl automated sequencer. In all cases, sequencing confirmed that the structure of the amplification product corresponded to the expected nucleotide sequence.

[0167] Example 4

[0168] Identification of mutations of tumor origin in the DNA of blood plasma

[0169] A comparative analysis of mutations was carried out in three DNA samples from the same patient with stage III malignant skin melanoma. The following sources were used as DNA sources: a) surgical material - the primary tumor - in the form of a paraffin block with formalin-fixed tissue; b) surgical material - a lymph node without signs of metastasis, but with suspected micrometastases by indirect signs (“watchdog” position of the node, increased size, abnormal structure of the stroma) - also in the form of a paraffin block with fixed formalin tissue; c) blood taken from the patient before surgery.

[0170] Samples presented as paraffin blocks were first subjected to morphological control. According to the results of morphological control, both samples were found to satisfy the criteria for use in this experiment and were used to isolate DNA without additional dissection procedures. Four glass products were made from each of the blocks. Used slides coated with poly-L-lysine (ApexLab, Moscow). For each glass slide, 2 sections were applied with a thickness of 6 μm and an area of 0.9 to 1.3 cm2. Slices were made on a rotational microtome RM5230 (Leica, Germany) using disposable blades; Before starting work with each new block, the working surfaces of the microtome were twice wiped with 96% ethanol; the first two slices from each block were not used (discarded) to exclude cross-contamination of the samples. To attach the slices to the poly-L-lysine coating, the slides, after applying the slices, were heated in a dry heat oven at a temperature of 56 ° C for 3 hours. Painting and conclusion of sections under coverslips were not made. DNA was isolated from attached sections using the QuickExtract FFPE DNA Extraction Kit (Epicentre, USA) in accordance with the manufacturer's instructions with slight modifications: after incubation at +90 C, the tubes with the DNA solution were placed at +4 C for 2 minutes, after which was centrifuged in a microcentrifuge (SM-50, BioSan, Latvia) at maximum speed for 10 minutes. A ring of concentrated paraffin formed above the solution was carefully punctured in the middle with a 300-liter disposable plastic tip for an automatic dispenser, after which the solution was taken through a puncture using a single-channel automatic mechanical dispenser and a similar 200-microliter tip; the solution was selected as carefully as possible, being careful not to trap the sediment at the bottom of the tube. The resulting DNA solution was stored at -20 C until further use.

[0171] A blood sample was taken by puncture of the ulnar vein into a 10 ml Vacutainer tube (Beckton Dickinson, USA) containing 1.1 ml of K3EDTA preservative. Blood was thoroughly mixed with a preservative and placed at +4 C. Not later than half an hour later, blood plasma was separated from blood cells using the standard protocol (described, in particular, in Appendix 2 to instructions for Insider kits, Evrogen CJSC, Moscow) . Plasma and blood cells were stored at -80 ° C until DNA was isolated. DNA from plasma was isolated using the QiaAMP Circulating Nucleic Acids Kit reagent kit (Qiagen, USA, cat. ID 55114) in accordance with the manufacturer's instructions. Final elution was performed twice, each time 11 μl of the elution buffer included in the reagent kit was used. The DNA solution was stored at -20 ° C until further use.

[0172] DNA samples were subjected to enzymatic fragmentation using the dsDNA Fragmentase reagent kit (New England Biolabs, USA) according to the manufacturer's instructions, the fragmentation results were monitored by ethidium bromide stained gel electrophoresis. Fragmentation was carried out until dominant fragments from 100 to 150 bp in length appeared. DNA concentration was measured using an XY-Detect reagent kit (Synthol, Russia, cat. No. HG-402) and a panel of DNA calibrators (at a concentration of 50, 10 and 1 ng / μl; Isogen Laboratory, Russia, cat. No. R0010) in according to manufacturers instructions.

[0173] The mutational status of the BRAF gene in samples of Τ (DNA from tumor tissue), LN (DNA from lymph node) and Ρ (DNA from blood plasma) was analyzed by standard and mutational-specific real-time PCR using the Insider B reagent kit -Raf (Eurogen, Russia) followed by Sanger sequencing. It was shown that mutation c is present in sample Τ. 1799 T → A (p. Val600Glu) in the 15th exon of the BRAF gene in about 45% of copies of the gene, in sample LN - in about 10% of copies, and it was not found in sample ((the selectivity of analysis for this sample was 1: 80).

[0174] Samples T, LN, and Ρ were used as a matrix for barcoding target fragments of the KRAS, NRAS, HRAS, ARAF, BRAF, CRAF, PIK3CA, and Akt1 genes. At the same time, two controls were also carried out through all stages of the barcoding reactions - positive control of PCR (30 ng of fragmented DNA from the Raji cell line) and negative control of PCR (NTC - no template control - control without DNA). Barcoding using linear PCR, purification and subsequent amplification of the target fragments was carried out as described in Example 3. Used sets of primers included in group B in Table. 2, obtained as described in example 3 (mixed in non-equimolar amounts), and primers for amplification of the internal control - IDH1 gene fragment. Linear PCR was performed for 5 cycles in duplicate, after treatment with an exonuclease and purification of the reaction product, the products of two replicates of linear PCR were combined and introduced into the reaction mixture for PCR amplification, which was carried out in the following mode: preliminary denaturation at 95 ° C for 5 min; 28 cycles: 95 ° C - 15 sec; 62 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes The PCR amplification product 1 was diluted 40 times and 1 μl of the diluted product was added to 50 μl of the reaction mixture for PCR with outflow (primers with 5 different index sequences were used for the three test samples, positive control and negative PCR control), which carried out in the mode: preliminary denaturation at 95 ° C for 5 min; 17 cycles: 95 ° C - 15 sec; 58 ° C - 20 sec; 72 ° C - 30 sec; completion of the ends of the molecules at 72 ° C for 2 minutes The amplification efficiency was monitored using agarose gel electrophoresis in TAE buffer (40 mM Tris acetate, pH 7.8-8.0; 1 mM EDTA) stained with ethidium bromide. PCR products of different samples were purified using a PCR product purification kit (Qiagen, Germany), the product concentration was measured on a Qubit 2.0 fluorimeter (Life Technologies, USA) using a Quant-IT dsDNA BR Assay Kit (Life Technologies, United States, Cat. ID Q32853) according to the manufacturer's instructions. 50 ng of amplification products of samples Τ and Raji (positive PCR control) and the entire volume of amplification products of samples LN, Ρ and NTC (negative PCR control) were combined (the total weight of the resulting PCR product mixture was 860 ng) and sequenced on a HiSeq2000 (Illumina) instrument using HiSeq PE Cluster Kit v3 cBot kits (Illumina, Cat. No. PE-301-3001) and HiSeq SBS Kit v.3 (Illumina, Cat. No. FC-301-2003) in accordance with the manufacturer's instructions. Paired (double-sided) readings with a length of 105 bases were obtained.

[0175] The results of massive sequencing were automatically sorted into samples according to index sequences using the CASAVA v.2.3.1 program (Illumina) and used to analyze mutations in the barcoded and amplified regions of the human genome. At the first stage of data analysis, data quality was evaluated according to a standard protocol using fastqc software with further filtering using flexbar software. Further analysis was performed using the MIGEC strategy (Shugay et al. Nat Meth. 2014; 11 (6): 653-5). The reliability of each individual reading was assessed, molecular barcodes were distinguished, the descendants of one source molecule were grouped, the consensus sequence was assembled within each “family” of molecules, the obtained consensus sequences were aligned to reference sequences, and secondary corrections of rare errors that accidentally dominated inside the individual “families” of molecules , and output the results in a table form.

[0176] The results obtained for exon 15 of the BRAF gene are presented in Table. 5. In the other studied regions, not a single “family” with any mutation was detected in any of the 5 samples.

[0177] Thus, it was possible to identify a small percentage of mutant DNA molecules of tumor origin in the blood plasma DNA of a patient with a tumor having an appropriate mutation with a selectivity of more than 1: 140. It is also important to note that low-represented molecules with a mutation would be indistinguishable from PCR and sequencing errors in the case of standard analysis, and it was possible to distinguish one from the other precisely thanks to the use of an analytical approach based on a preliminary grouping of reads into “families” based on individual molecular barcodes.

> SEQ ID NO: 1

aaatgagatctactgttttcctttacttactacacctcagATATATTTCTTCATGAAGACCTCACAGTAAAAATAGGTGATTTTTGGTCTAGCTACAGTGAAATCTCGAT

> SEQ ID NO: 2

TCCCCTTCCCTGCCCCAGCCAATGCCCCCCTACAGCGCATCCGCTCCACGTCCACTCCCAACGTCCATATGGTCAGCACCACGGCCCCATGGACTCCAACCTCATCCAGgttggtgctgtgggggacaatgccggggacac

> SEQ ID NO: 3

CCTCACGCCTTCACCTTTAACACCTCCAGTCCCTCATCTGAAGGTTCCCTCTCCCAGAGGCAGAGGTCGACATCCACACCTAATGTCCACATGGTCAGCACCACCCTGCCTGTGGAC

> SEQ ID NO: 4

acagAAACCCATGTATGAAGTACAGTGGAAGGTTGTTGAGGAGATAAATGGAAACAATTATGTTTACATAGACCCAACACAACTTCCTTATGATCACAAATGGGAGTTTCCCAGAAACAGGCTGAGTTTTGgtcagt

> SEQ ID NO: 5

AGCTGGTGGCGTAGGCAAGAGTGCCTTGACGATACAGCTAATTCAGAATCATTTTTGTGGACGAATATGATCCAACAATAGAGGtaaatcttgttttaatatgcatattactggt

> SEQ ID NO: 6

gtgtttctcccttctcagGATTCCTACAGGAAGCAAGTAGTAATTGATGGAGAAACCTGTCTCTTGGATATTCTCGACACAGCAGGTCAAGAGGAGTACAGTGC

> SEQ ID NO: 7

tan

> SEQ ID NO: 8

TGCTGGTGTGAAATGACTGAGTACAAACTGGTGGTGGTTGGAGCAGGTGGTGTTGGGAAAAGCGCACTGACAATCCAGCTAATCCAGAACCACTTTGTAGATGAATATATGATCCCACCATAGAGgtgaggcccc

> SEQ ID NO: 9

CTGTTTGTTGGACATACTGGATACAGCTGGACAAGAAGAGTACAGTGCCATGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCTCTGTGTATTTGCCATCAATAATAG

> SEQ ID NO: 10

GTGGGAAACAAGTGTGATTTGCCAACAAGGACAGTTGATACAAAACAAGCCCACGAACTGGCCAAGAGTTACGGGATTCCATTCATTGAAACCTCAGCCAAGACCAGACAGgta

> SEQ ID NO: 11

CCCGGGCCGCAGGCCCCTGAGGAGCGATGACGGAATATAAGCTGGTGGTGGTGGGCGCCGGCGGTGTGGGCAAGAGTGCGCTGACCATCCAGCTGATCCAGAACCATTTTGTGGACGAATACGACCC

> SEQ ID NO: 12

CCGGAAGCAGGTGGTCATTGATGGGGAGAGGTGCCTGTTGGACATCCTGGATACCGCCGGCCAGGAGGAGTACAGCGCCATGCGGGACCAGTACATGCGCACCGGGGAGAGGGC

> SEQ ID NO: 13

GATGACGTGCCCATGGTGCTGGTGGGGAACAAGTGTGACCTGGCTGCACGCACTGTGGAATCTCGGCAGGCTCAGGACCTCGCCCGAAGCTACGGCATCCCCTACATCGAGACCTCGGCCAAGACCCGGCAGg

> SEQ ID NO: 14

AACTTGGAGGCCTTGCAGAAGAAGCTGGAGGAGCTAGAGCTTGATGAGCAGCAGCGAAAGCGCCTTGAGGCCTTTCTTACCCAGAAGCAGAAGGTGGGAGAACTGAAGGATGACGACTTTGAGAAGATCAGTGAGCGGGGGT

> SEQ ID NO: 15

CATGTGCTCCATGCAGATACTGATCTCGCCATCGCTGTAGAACGCACCATAGAAGCCCACGATGTACGGAGAGTTGCACTCATGCAGAACCTGCAGCTCCCTTATGATCTGGTTCCGGATTGCGGGTTGATC

> SEQ ID NO: 16

GTGGGGAGATCAAGCTCTGTGACTTTGGGGTCAGCGGGCAGCTCATCGACTCCATGGCCAACTCCTTCGTGGGCACAAGGTCCTACATGTCGgtatgaacagaagtttccattgcttgagctt

> SEQ ID NO: 17

ggccacctgcgcagagcctgcactcactccttgtgtctccccctagGACGGCGGCTCCCTGGACCAGGTGCTGAAAGAGGCCAAGAGGATTCCCGAGGAGATCCTGGGGAAAGTCAGCATCGCGgtgagtccaccgg

> SEQ ID NO: 18

CCTCCAACATCCTCGTGAACTCTAGAGGGGGAGATCAAGCTGTGTGACTTCGGGGTGAGCGGCCAGCTCATCGACTCCATGGCCAACTCCTTCGT

> SEQ ID NO: 19

acctttctctttatagCTCTGGACTTATTGGACAAAATGTTGACATTCAACCCACACAAGAGGATTGAAGTAGAACAGGCTCTGGCCCACCCATATCTGGAGCAGTATTACGACCCGAGTGACGAGgtaagagctca

> SEQ ID NO: 20

TAAGGGAAAATGACAAAGAACAGCTCAAAGCAATTTCTACACGAGATCCTCTCTCTCTGAAATCACTGAGCAGGAGAAAGATTTTCTATGGAGTCACAGGTAAGTGCTAAAAA

> SEQ ID NO: 21

ATTCGAAAGACCCTAGCCTTAGATAAAACTGAGCAAGAGGCTTTGGAGTATTTCATGAAACAAATGAATGATGCACATCATGGTGGCTGGACAACAAAAAGGATTGGATCTTCCACACAATTAAACAGCAT

> SEQ ID NO: 22

TCATTCTTGAGGAGGAAGTAGCGTGGCCGCCAGGTCTTGATGTACTCCCctacagacgtgc

> SEQ ID NO: 23

aatattttccctaagtttgtaagtagtgctatatttatgttgactttttagttttactttatatgttattaatatgagtattgttaaccttgcagAATGGTCGATGTAGGGGGCCAAAGGTCAGAGAGAAGAAAATGGATACACTGCTTTAGATGTATTAGTATTAGATCATTAGTATTAGATATTAGATATTAGATATTAGATATTAGATAGTATTAGATATTAGATAGTATTAGATAGTATTAGATAGTATTAGATAGTAGTTAGATAGTAGTTAGATAGTAGTTAGATAGTTAGATAGTAGTAGTAGTAGTAGTAGTAGTAGGTAGTAGTAGTAGTAGTAGTAGGTAGTAGTAGTAGTAGGTAGTAGGTAGTAGGTAGTAGTAGTAGGTAGTAGTAGTAGGTAGTAGGTA

> SEQ ID NO: 24

ggccttggggcgccaggtggctgagtcctggcgctgtgctcctttcagGATGGTGGATGTGGGGGGCCAGCGGTCGGAGCGGAGGAAGTGGATCCACTGCTTTGAGAACGTGACATCCATCATGTTTCTCGTCGCG

> SEQ ID NO: 25

accttttttctctttctctttagAGCTGTAGGTAAAACTTGCCTACTGATCAGTTACACAACCAATGCATTTCCTGGAGAATATATCCCTACTGTgtaagtatcttaaattgggaatta

> SEQ ID NO: 26

TGATAATTTCAGGAAACAAAAATTTGTGCTATGCAAATACAATAAACTGGAAAAAACTGTTTGGGACCTCCGGTCAGAAAACCAAAATTATAAGCAACAGAGGTGAAAACAGCTGCA

> SEQ ID NO: 27

tacCTTATACACCGTGCCGAACGCACCGGAGCCCAGCACTTTGATCTTTTTGAATTCAGTTTCCTTCAAGATCCTCAAGAGAGCTTGGTTGGGAGCTTCTCCACTGGGTGTAAGAGGCTCCACAAGctgggg

> SEQ ID NO: 28

gtcttccttctctctctgtcatagGGACTCTGGATCCCAGAAGGTGAGAAAGTTAAAATTCCCGTCGCTATCAAGGAATTAAGAGAAGCAACATCTCCGAAAGCCAACAAGGAAATCCTCGATgtgagttt

> SEQ ID NO: 29

gtgcctctccctccctccagGAAGCCTACGTGATGGCCAGCGTGGACAACCCCCACGTGTGCCGCCTGCTGGGCATCTGCCTCACCTCCACCGTGCAGCTCATCACGCAGCTCATGCCCTTCGGCTGCCTCC

> SEQ ID NO: 30

ccttccctgattacCTTTGCGATCTGCACACACCAGTTGAGCAGGTACTGGGAGAGAATATTGTCTTTGTGTTCCCGGACATAGTCCAGGAGGCAGCCGAAGGGCATGAGCTGCGTGATGAGCTGC

> SEQ ID NO: 31

ccttacTTTGCCTCCTTCTGCATGGTATTCTTTCTCTTCCGCACCCAGCAGTTTGGCCAGCCCAAAATCTGTGATCTTGACATGCTGCGGTGTTTTCACCAGTACGTTCCTGGCGAGGGGAGGTAGCGTAGCGAGTAGCTAGTAGCTAGTAGCTAG

> SEQ ID NO: 32

tccttagACAACTACCTTTCTACGGACGTGGGATCCTGCACCCTCGTCTGCCCCCTGCACAACCAAGAGGTGACAGCAGAGGATGGAACACAGCGGTGTGAGAAGTGCAGCAAGCCCTGTGCCCGAGgtacccgactccccggcc

> SEQ ID NO: 33

ccccagCTGGTGGAGCCGCTGACACCTAGCGGAGCGATGCCCAACCAGGCGCAGATGCGGATCCTGAAAGAGACGGAGCTGAGGAAGGTGAAGGTGCTTGGATCTGGCGCTTT

> SEQ ID NO: 34

TGCTTGGATCTGGCGCTTTTTGGCACAGTCTACAAGgtcagggccaggtcctggggtgggcggccccagagatggggcggtgcctgg

> SEQ ID NO: 35

ctgcccagGGCATCTGGATCCCTGATGGGGAGAATGTGAAAATTCCAGTGGCCATCAAAGTGTTGAGGGAAAACACATCCCCCAAAGCCAACAAAGAAATCTTAGACgtaagcccctccaccctctcctgc

> SEQ ID NO: 36

tacccttgtccccagGAAGCATACGTGATGGCTGGTGTGGGCTCCCCATATGTCTCCCGCCTTCTGGGCATCTGCCTGACATCCACGGTGCAGCTGGTGACACAGCTTATG

> SEQ ID NO: 37

ATGCCCTATGGCTGCCTCTTAGACCATGTCCGGGAAAACCGCGGACGCCTGGGCTCCCAGGACCTGCTGAACTGGTGTATGCAGATTGCCAAGgtatgcacctg

> SEQ ID NO: 38

atgggtgcttcccattccagGGGATGAGCTACCTGGAGGATGTGCGGCTCGTACACAGGGACTTGGCCGCTCGGAACGTGCTGGTCAAGAGTCCCAACCATGTCAAAATTACAGACTTC

> SEQ ID NO: 39

AAATTACAGACTTCGGGCTGGCTCGGCTGCTGGACATTGACGAGACAGAGTACCATGCAGATGGGGGCAAGgttaggtgaaggaccaaggagcagaggaggctgg

> SEQ ID NO: 40

CGGCGATGCTGAGAACCAATACCAGACACTGTACAAGCTCTACGAGAGGTGTGAGGTGGTGATGGGGAACCTTGAGATTGTGCTCACGGGAC

> SEQ ID NO: 41

CTATGTCCTCGTGGCCATGAATGAATTCTCTACTCTACCATTGCCCAACCTCCGCGTGGTGCGAGGGACCCAGGTCTACGATGGGAAGTTTGCCATCTTCGTCATGTTGAACTATAACACCAACTCCCC

> SEQ ID NO: 42

ttctctccttccatagTGACCAAGACCATCTGTGCTCCTCAGTGTAATGGTCACTGCTTTGGGCCCCACACCCCAACCAGTGCTGCCATGATGAGTGTGCCGGGGGCTGCTCAGGCCCTCAGGACACAGACTGCTTgtg

> SEQ ID NO: 43

CCACAGCCTCTTGTCTACAACAAGCTAACTTTCCAGCTGGAACCCAATCCCCACACCAAGTATCAGTATGGAGGAGTTTGTGTAGCCAGCTGTCCCCgtaagtgtctgaggggaagga

> SEQ ID NO: 44

tcatctctaatggtgtcctcctcctcttccctagATAACTTTGTGGTGGATCAAACATCCTGTGTCAGGGCCTGTCCTCCTGACAAGATGGAAGTAGATAAAAATGGGCTCAAGATGTGTGAGCCCCTTGTGGGGAGATGTGGGAGATGT

> SEQ ID NO: 45

GGGAACAGGCTCTGGGAGCCGCTTCCAGACTGTGGACTCGAGCAACATTGATGGATTTGTGAACTGCACCAAGATCCTGGGCAACCTGGACTTTCTGATCAC

> SEQ ID NO: 46

tacagGGAATGTACTACCTTGAGGAACATGGTATGGTGCATAGAAACCTGGCTGCCCGAAACGTGCTACTCAAGTCACCCAGTCAGGTTCAGGTGGCAGATTTTGGTGTGGCTGACCTGCTGCCTAGCATGATGATGAT

> SEQ ID NO: 47

ttcctgcaacagGTGTGACAGTTTGGGAGTTGATGACCTTCGGGGCAGAGCCCTATGCAGGGCTACGATTGGCTGAAGTACCAGACCTGCTAGAGAAGGGGGAGCGGTTGGCACAGCCCCAGATCTGCA

> SEQ ID NO: 48

ctgaatgttaacattaatgcttattttaccttttttttttttttaatcagATAACTTTGTGGTAGATTCCAGTTCTTGTGTGCGTGCCTGCCCTAGTTCCAAGATGGAAGTAGAAGAAAA

> SEQ ID NO: 49

AGATGGAAGTAGAAGAAAATGGGATTAAAATGTGTAAACCTTGCACTGACATTTGCCCAAAAGgtgagtggtagctcagattatttagttagaactgggcctatat

> SEQ ID NO: 50

tttctcacttccccctccttagTGGCCTGTCCTTGCTTATCCTCAAGCAACAGGGCATCACCTCTCTACAGTTCCAGTCCCTGAAGGAAATCAGCGCAGGAAACATCTATATTACTGACAACAGCAACCC

> SEQ ID NO: 51

TATTACTGACAACAGCAACCTGTGTTATTATCATACCATTAACTGGACAACACTCTCTTCAGCACAATCAACCAGAGAATAGTAATCCGGGACAACAGAAAAGCTGAAAATTGTAgtaagtacattactcagacatttaaaacatagaaacttaaattt

> SEQ ID NO: 52

tcctcatgtggctgatgttttcacagTGAATTTCGGGAGTTTGGAATGGCTCCATCTGTGTGGAGTGTGACCCCCAGTGTGAGAAGATGGAAGATGGCCTCCTCACATGCCATGGACCGgtaag

> SEQ ID NO: 53

atatttttcagGGAATGATGTACCTGGAAGAAAGACGACTCGTTCATCGGGATTTGGCAGCCCGTAATGTCTTAGTGAAATCTCCAAACCATGTGAAAATCACAGATTTTGG

> SEQ ID NO: 54

TGAAATCTCCAAACCATGTGAAAATCACAGATTTTGGGCTAGCCAGACTCTTGGAAGGAGATGAAAAAGAGTACAATGCTGATGGAGGAAAGgtgggtatcttttt

> SEQ ID NO: 55

tcaactctgtctccttcctcttcctacagTACTCCCCTGCCCTCAACAAGATGTTTTGCCAACTGGCCAAGACCTGCCCTGTGCAGCTGTGGGTTGATTCCACACCCCCCCGCCCGGCACCCGCGTCCGCGCCATGGC

> SEQ ID NO: 56

TTCCACACCCCCGCCCGGCACCCGCGTCCGCGCCATGGCCATCTACAAGCAGTCACAGCACATGACGGAGGTTGTGAGGCGCTGCCCCCACCATGAGCGCTGCTCAGATAGCGATGgtgagcagctggggc

> SEQ ID NO: 57

ctcactgattgctcttagGTCTGGCCCCTCCTCAGCATCTTATCCGAGTGGAAGGAAATTTGCGTGTGGAGTATTTGGATGACAGAAACACTTTTCGACATAGTGTGGTGGTGCCCTATGAGCCGCCTGAGgctggttgg

> SEQ ID NO: 58

ttatctcctagGTTGGCTCTGACTGTACCACCATCCACTACAACTACATGTGTAACAGTTCCTGCATGGGCGGCATGAACCGGAGGCCCATCCTCACCATCATCACACTGGAAGACTCCAGgtcaggagcca

> SEQ ID NO: 59

agtagTGGTAATCTACTGGGACGGAACAGCTTTGAGGTGCGTGTTTGTGCCTGTCCTGGGAGAGACCGGCGCACAGAGGAAGAGAATCTCCGCAAGAAAGGGGAGCCTCACCACGAGCTGCCCCCAGGGAGGAGTA

> SEQ ID NO: 60

ttgcctctttcctagCACTGCCCAACAACACCAGCTCCTCTCCCCAGCCAAAGAAGAAACCACTGGATGGAGAATATTTCACCCTTCAGgtactaagtctt

> SEQ ID NO: 61

atttatagCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTC

> SEQ ID NO: 62

CACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGGTAAGAGTATTATTTTTCAT

> SEQ ID NO: 63

TTGATCCCCATAAGCATGACGACCTATGATGATAGGTTTTACCCATCCACTCACAAGCCGGGGGATATTTTTGCAGATAATGGCTTCTCTGAAGACCGTGCCA

> SEQ ID NO: 64

GCAGCCGTCCTGGAGGCTGGTGTCCTGGGTAGCGGTCAGCACGCCCCCGTCTTCGTATGTGGTGACTCTCTCCCATGTGAAGCCCTCGGGGA

> SEQ ID NO: 65

AAACACTCGGCTGGGAGGCCTCCACCGAGACCCTGTACCCCGCTGACGGCGGCCTGGAAGGCAGAGCCGACATGGCCCTGAAGCTCGTGGGCGGGGGCCACCTGATCTGCAACTTGA

> SEQ ID NO: 66

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCATCTCACAATTGCCAGTTAAC

> SEQ ID NO: 67

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTATCTCCCCTCCCCGTATCTC

> SEQ ID NO: 68

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCTGACCCACACACACCCCCTCA

> SEQ ID NO: 69

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGGTCCTTCCTGTCCTCCTA

> SEQ ID NO: 70

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCGGAGAGACCTGCAAAGAGCC

> SEQ ID NO: 71

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNATGGGCTAGACACCACTCCAC

> SEQ ID NO: 72

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNGAGAGAGAGGTCGGCATTGT

> SEQ ID NO: 73

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGAGCCCTAACAGCCATGCT

> SEQ ID NO: 74

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTAGCTGGTGATGTTCCTCCC

> SEQ ID NO: 75

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNANAACTACTTTCTTCCCCTATACC

> SEQ ID NO: 76

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNTATCTTGTTTTGAGCTTGTTTG

> SEQ ID NO: 77

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGTCCAAAGAAGAATGGGAAA

> SEQ ID NO: 78

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTTGACGCTTGTTTGCTTACACC

> SEQ ID NO: 79

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTTTTCAAGCAAGATTGCTCTC

> SEQ ID NO: 80

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGCTGTGACTGCTTGTAGATG

> SEQ ID NO: 81

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNAGGGTCCCCAGGCCTCTGATTC

> SEQ ID NO: 82

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGCTTCTCTTTTCCTATCCTG

> SEQ ID NO: 83

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGAGGCCTGTGCCAGGGACCT

> SEQ ID NO: 84

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNACCATGCGAAGCCACACTGAC

> SEQ ID NO: 85

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNN CTGGCTGACCTAAAGCCACCT

> SEQ ID NO: 86

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGACAGGAACTGCAGCTGGC

> SEQ ID NO: 87

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGAACTGCCGACCACACCCCCCT

> SEQ ID NO: 88

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGTCTCCCATACCCTCTCAGCG

> SEQ ID NO: 89

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGCCCTCCCAGAAGGTCTAC

> SEQ ID NO: 90

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGGATTCGAGAAGTGACAGG

> SEQ ID NO: 91

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGATCTACTATTGTTGATCATTGT

> SEQ ID NO: 92

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNTCTTACATTTGCAGCCTGTGA

> SEQ ID NO: 93

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGCCAGTGACTAGTCCATGTC

> SEQ ID NO: 94

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNCTTAAAAGTGAATTAAAAACCTTGGT

> SEQ ID NO: 95

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGAATGCGTTTCATGGTTCCGT

> SEQ ID NO: 96

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCTGAATATCATTAAGGAACTTG

> SEQ ID NO: 97

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCCAGCCCTGTCGTCTCTCCA

> SEQ ID NO: 98

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGCCTCATCTTGGGCCTGTG

> SEQ ID NO: 99

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNATTCACTTTTATCACCTTTCC

> SEQ ID NO: 100

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCATAATGCTTGCTCTGATAGGA

> SEQ ID NO: 101

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNACTGTGACCCGGAGCACT

> SEQ ID NO: 102

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTTCTCAGCACAGATATTCTACA

> SEQ ID NO: 103

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCTATTTTTCCCTTTCTCCCC

> SEQ ID NO: 104

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTATAAACTTGTGGTAGTTGG

> SEQ ID NO: 105

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGCACTGTAATAATCCAGACT

> SEQ ID NO: 106

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCAGTGTTACTTACCTGTCTTG

> SEQ ID NO: 107

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNTTACTGGTTTCCAACAGGTTCT

> SEQ ID NO: 108

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNAAGTGGTTATAGATGGTGAAAC

> SEQ ID NO: 109

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNACACAAATGCTGAAAGCTGTACCA

> SEQ ID NO: 110

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGCCAGGCTCACCTCTATAGTG

> SEQ ID NO: 111

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGATGGCAAACACACACAGAGAA

> SEQ ID NO: 112

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGGTGGAGAGCTGCCTCA

> SEQ ID NO: 113

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGGAAAATGACAAAGAACAGC

> SEQ ID NO: 114

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNTCTTTTGATGACATTGCATAC

> SEQ ID NO: 115

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGCCAACCCCCCACAAATCTGAA

> SEQ ID NO: 116

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNAGGAGATAAGTGATGGAGATG

> SEQ ID NO: 117

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCATTCTGACTTTCAGTAAGGCA

> SEQ ID NO: 118

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNTCCCCACTTTGGAACAGGACC

> SEQ ID NO: 119

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGCTGAGAGGGTGTCACATAC

> SEQ ID NO: 120

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCCTCTCCCTGACCGTACAAG

> SEQ ID NO: 121

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGGGCGCGATGTGGGTCTG

> SEQ ID NO: 122

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGCCGCCTCCAGATGTGAAGC

> SEQ ID NO: 123

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCTTCTGCTCTCACTACTGCAAA

> SEQ ID NO: 124

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNGGATGATCATCGTCATTCAAGAG

> SEQ ID NO: 125

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCCGTCCTGGGATTGCAGATTG

> SEQ ID NO: 126

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTACATTCATGTTGACTAAGCA

> SEQ ID NO: 127

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNCTACTAATGCTAATACTGTTTCGT

> SEQ ID NO: 128

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCATTATTGCCAACATGACTTAC

> SEQ ID NO: 129

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNTGATCTTGACGTTGTAGATGAG

> SEQ ID NO: 130

ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNCGGCCCTGTGATGCAGAAGA

> SEQ ID NO: 131

GAGGTTCAGAGCCATGGACC

> SEQ ID NO: 132

ACGTGTGCCGCCTGCTGGG

> SEQ ID NO: 133

CCTGGCCCTGACCTTGTAGAC

> SEQ ID NO: 134

CCCACGCTCTTCTCACTCATA

> SEQ ID NO: 135

CATCTGCCTGACATCCACGGT

> SEQ ID NO: 136

GGAACGTGCTGGTCAAGAGTC

> SEQ ID NO: 137

TCCTGGGACTCTGAATGGC

> SEQ ID NO: 138

AATTTCAGAACCCAGGCAATG

> SEQ ID NO: 139

GAAAGGCTGTCTCTTTACAACT

> SEQ ID NO: 140

CTTTGTGTCTCCTCACCTTG

> SEQ ID NO: 141

TTTGGGCAAATGTCAGTGCAAG

> SEQ ID NO: 142

CAGTTCCAGTCCCTGAAGGA

> SEQ ID NO: 143

TAAATAAGCCAACACACCACAG

> SEQ ID NO: 144

GACTCGTTCATCGGGATTTG

> SEQ ID NO: 145

GTTGCTTTATCTGTTCACTTG

> SEQ ID NO: 146

CACCCGGAGGGCCACTGACAAC

> SEQ ID NO: 147

CTCCACCGCTTCTTGTCCT

> SEQ ID NO: 148

AGGGCTGAGGTGACCCTTGT

> SEQ ID NO: 149

AGCCAATATTGTCTTTGTGTTCC

> SEQ ID NO: 150

ATGATCTGTCCCTCACAGCA

> SEQ ID NO: 151

CCAGGGTATGTGGCTACATG

> SEQ ID NO: 152

CCTGAAAGAGACGGAGCTGAG

> SEQ ID NO: 153

TCCCGGACATGGTCTAAGAG

> SEQ ID NO: 154

CTCGTCAATGTCCAGCAGCC

> SEQ ID NO: 155

GGTGAGCTGAGTCAAGCGG

> SEQ ID NO: 156

CGGCACTTCAATGACAGTGGA

> SEQ ID NO: 157

GGGAAGGCAGGATCTCTAAC

> SEQ ID NO: 158

CAGCAGGTAACTCACACTTGA

> SEQ ID NO: 159

GATTCCAGTTCTTGTGTGCGT

> SEQ ID NO: 160

GCTGAAGAGTGTTGTCCAGT

> SEQ ID NO: 161

CTTTCCTCCATCAGCATTGTA

> SEQ ID NO: 162

CTCAACAAGATGTTTTGCCAAC

> SEQ ID NO: 163

GGCAAGCAGAGGCTGGGGCAC

> SEQ ID NO: 164

ACGGCATTTTGAGTGTTAGAC

> SEQ ID NO: 165

CAACTGTTCAAACTGATGGGA

> SEQ ID NO: 166

GCCAACAGGGATGGCTCTA

> SEQ ID NO: 167

CAAGGTGCCCTATTACCTCA

> SEQ ID NO: 168

TACCCAAAAAGGTGACATGG

> SEQ ID NO: 169

TGAAAATGGTCAGAGAAACCT

> SEQ ID NO: 170

CTCCCCAGTCCTCATGTACT

> SEQ ID NO: 171

AAAGAGTTAAGGACTCTGAAGA

> SEQ ID NO: 172

CAAGTGAGAGACAGGATCAGG

> SEQ ID NO: 173

ACCTGTAGAGGTTAATATCCG

> SEQ ID NO: 174

AGGGAGCAGATTAAGCGAGT

> SEQ ID NO: 175

GGCAGGTGGGGCAGGAGAC

> SEQ ID NO: 176

TCCCTGAGCCTTGTCCTCCT

> SEQ ID NO: 177

TCTCGCTTTCCACCTCTCAG

> SEQ ID NO: 178

AACATGCTGAGATCAGCCAGAT

> SEQ ID NO: 179

AGTGTGGAATCCAGAGTGAGC

> SEQ ID NO: 180

TCTGACGGGTAGAGTGTGC

> SEQ ID NO: 181

CAAATAAAGGACCCATTAGAACC

> SEQ ID NO: 182

CTTACCTGGACTCTGGAATC

> SEQ ID NO: 183

AGGCCAGAAGGCTTGTGGG

> SEQ ID NO: 184

CCCTCTTTCTTTCATAAAACCTC

> SEQ ID NO: 185

TTCTCTTCCCTGCAGATGTCA

> SEQ ID NO: 186

GGGAGTGGGGGGACGCATC

> SEQ ID NO: 187

CAGCGGGGACTCACAGCCAT

> SEQ ID NO: 188

ATCTATGTCCCTGAAGCAGC

> SEQ ID NO: 189

CTTACCTCATTGTCTGACTCC

> SEQ ID NO: 190

CTCGTTGTCCGACTCCACCA

> SEQ ID NO: 191

CATATGAGGATTAAAGATGGCTT

> SEQ ID NO: 192

CATCCTCTTCCTCAGGATTGC

> SEQ ID NO: 193

ATCACCAAATGGCACCATAC

> SEQ ID NO: 194

ACCTTCATCAACCACACCCA

> SEQ ID NO: 195

GCATCTTGAGGTTCTTAGCG

> SEQ ID NO: 199

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCCATGGACCCCCACACACAG

> SEQ ID NO: 200

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCATCTGCCTCACCTCCACCGT

> SEQ ID NO: 201

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGACCTTGTAGACTGTGCCA

> SEQ ID NO: 202

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCACTCATATCCTCCTCTTT

> SEQ ID NO: 203

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGCAGCTGGTGACACAGCTT

> SEQ ID NO: 204

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAAGAGTCCCAACCATGTCA

> SEQ ID NO: 205

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGAATGGCCTGAGTGTGAC

> SEQ ID NO: 206

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCAGGCAATGGAAAGGGTA

> SEQ ID NO: 207

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTTACAACTTCTTACCATCTC

> SEQ ID NO: 208

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTGGCCTCACTGTATAGCA

> SEQ ID NO: 209

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGTTTACACATTTTAATCCCA

> SEQ ID NO: 210

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAATCAGCGCAGGAAACATCTA

> SEQ ID NO: 211

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACACCACAGATGTCTTCAGG

> SEQ ID NO: 212

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGCAGCCCGTAATGTCTTAG

> SEQ ID NO: 213

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTCACTTGTGCCCTGACTT

> SEQ ID NO: 214

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTAACCCCTCCTCCCAGAGAC

> SEQ ID NO: 215

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACCGCTTCTTGTCCTGCTTG

> SEQ ID NO: 216

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCTCTGTGTTCTTGTCCC

> SEQ ID NO: 217

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAGCCGAAGGGCCATGAGCT

> SEQ ID NO: 218

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCTCACAGCAGGGTCTTC

> SEQ ID NO: 219

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTGGCTACATGTTCCTGATC

> SEQ ID NO: 220

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAGCTGAGGAAGGTGAAGG

> SEQ ID NO: 221

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAGAGGCAGCCATAGGG

> SEQ ID NO: 222

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCAGCCGAGCCAGCCC

> SEQ ID NO: 223

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTGGCGCAGAGCGTGGCT

> SEQ ID NO: 224

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGCCTGTGTACCTCGCTGT

> SEQ ID NO: 225

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCTCTAACCATTGAGGCCG

> SEQ ID NO: 226

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCACACTTGACCATCACCA

> SEQ ID NO: 227

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCGTGCCTGCCCTAGTTCCA

> SEQ ID NO: 228

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGTTAATGGTATGATAATAACACA

> SEQ ID NO: 229

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTTCCAAGAGTCTGGCTAGC

> SEQ ID NO: 230

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTGCAGCTGTGGGTTGA

> SEQ ID NO: 231

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCAGTGTGCAGGGTGGCAAG

> SEQ ID NO: 232

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCACTTGATAAGAGGTCCC

> SEQ ID NO: 233

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAACTGATGGGACCCACTCC

> SEQ ID NO: 234

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCTACCCTCTGCCCTGTG

> SEQ ID NO: 235

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATTACCTCAATCATCCTGCT

> SEQ ID NO: 236

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCATGGAAAGCCCCTGTTTCAT

> SEQ ID NO: 237

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTATCAAAGAATGGTCCTGC

> SEQ ID NO: 238

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCATGTACTGGTCCCTCATT

> SEQ ID NO: 239

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACTCTGAAGATGTACCTATG

> SEQ ID NO: 240

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGGTCAGCGGGCTACCACTG

> SEQ ID NO: 241

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTAATATCCGCAAATGACTTG

> SEQ ID NO: 242

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGATGATGTACCTATGGTGCTA

> SEQ ID NO: 243

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGGAGACCCTGTAGGAGGAC

> SEQ ID NO: 244

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTCCTCCTGCAGGATTCCTA

> SEQ ID NO: 245

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAAACGGGGTGAAGGACTCG

> SEQ ID NO: 246

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAAAAGAAACAGAGAATCTCCA

> SEQ ID NO: 247

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGTTATCTTTTCAGTTCAATGC

> SEQ ID NO: 248

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAGAGTGTGCGTGGCTCTCA

> SEQ ID NO: 249

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAAACAGAAAGCGGTGACTTAC

> SEQ ID NO: 250

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATCCATTCTGGTGCCACTAC

> SEQ ID NO: 251

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTGAACACCACACCGCCATTG

> SEQ ID NO: 252

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCTCTTTCTTCCACCTTTCTCC

> SEQ ID NO: 253

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCAACATCCTAGTCAACTCCC

> SEQ ID NO: 254

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGACGCATCCGTGGCTTCCC

> SEQ ID NO: 255

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCATGTAGGAGCGCGTGCCC

> SEQ ID NO: 256

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGAAGCAGCAGCCAGGAACA

> SEQ ID NO: 257

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTATATTCACTAAGCGCTACTAGA

> SEQ ID NO: 258

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTACTTGGTCGTATTCGCTGAGG

> SEQ ID NO: 259

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAACACAAACAGGTTAATTCCCA

> SEQ ID NO: 260

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTGGATTGCCTTTACCACTCAGA

> SEQ ID NO: 261

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCACCATACGAAATATTCTGGG

> SEQ ID NO: 262

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCGACTTCTTTAAGCAGTCCT

> SEQ ID NO: 263

GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTCTTGGATCTGTATGTGGTCT

> SEQ ID NO: 264

CAAGCAGAAGACGGCATACG AGATTGGTCAGTGACTGGAGTTCAGACGTGT

> SEQ ID NO: 265

CAAGCAGAAGACGGCATACGAGAGTGACTGGAGTTCAGACGTGT

> SEQ ID NO: 266

CAAGCAGAAGACGGCATACGAGATCGTGATGTGACTGGAGTTCAGACGTGT

> SEQ ID NO: 267

CAAGCAGAAGACGGCATACGAGATACATCGGTGACTGGAGTTCAGACGTGT

Claims (13)

1. A method for detecting mutations in target DNA sequences, including: (a) molecular barcoding of a pool of target DNA molecules in a DNA sample by introducing a random sequence into the target DNA molecule sequences by multiplex linear PCR in the presence of barcoding primers carrying gene-specific sequences at the 3'-end complementary to the target DNA sequences followed by a random sequence ( barcode), and the adapter sequence at the 5'-end; (b) purification of the product of the barcoding reaction from residues of the barcoding primer, including incubation with exonuclease I and further purification using gel filtration; (c) amplification of the pool of target molecules using multiplex PCR with a primer essentially similar to the adapter sequence of the barcoding primer and counter gene-specific primers for the target regions; (d) introducing sequence adapters into the PCR product using PCR with the release to the outside in the presence of buried internal gene-specific primers and sequencing the specified PCR product; (e) the identification of mutations in the target DNA according to sequencing data. 2. A method for detecting mutations in target DNA sequences selected from SEQ ID No: 1 - SEQ ID No: 62, including: (a) molecular barcoding of a pool of target DNA molecules in a DNA sample by introducing a random sequence in the sequence of target DNA molecules by multiplex linear PCR in the presence of barcoding primers selected from the target sequences selected from the group SEQ ID NOs: 66-127 (b) purification of the product of the barcoding reaction from residues of the barcoding primer, including incubation with exonuclease I and further purification using gel filtration; (c) amplification of the pool of target molecules using multiplex PCR with a primer essentially similar to the adapter sequence of the barcoding primer and counter gene-specific primers for the target regions selected from SEQ ID NOs: 131-192; (d) introducing sequence adapters into the PCR product by PCR going out in the presence of buried internal gene-specific primers selected from SEQ ID NOs: 199-260, and sequencing said PCR product; (e) the identification of mutations in the target DNA according to sequencing data. 3. A set of oligonucleotide primers for implementing the method according to claim 1 or 2, selected from groups A, B, C or D shown in table 2, where the specified set includes a set of primers for barcoding, a set of REV primers for amplification and a set of BRIDGE primers for introducing sequencing adapters.

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