KR20160082715A - Detection method of gene deletion based on next-generation sequencing - Google Patents

Abstract

The present invention relates to a method for detecting a deletion gene cluster based on next-generation sequence analysis. The gene deletion develops because of diseases, and it is important to find a gene deletion portion in the analysis of sequencing. However, conventional variant calling sequence analysis has limitations on detection of deletion gene. A read distribution sequence analysis method of the present invention is expected to be greatly used in diagnosis and monitoring of diseases by accurately discriminating and diagnosing wild type, hetero deletion type, and homo deletion type genes based on next-generation sequence analysis.

Description

[0001] The present invention relates to a method for detecting a deletion gene group based on a next-generation sequencing method,

The present invention relates to a method for detecting a deletion gene group based on a next-generation sequencing method.

Since the beginning of the human genome project, much research has been done on the genes responsible for the disease, and sequencing techniques for identifying the nucleotide sequences of the genes are being developed. In recent years, next generation sequencing (NGS), which produces large sequences of short sequences due to low cost and rapid data production, is rapidly replacing traditional Sanger sequencing. With the development of the next generation sequencing technology, the cost of producing short sequences has become less than half of what it used to be, and the availability of applications for disease diagnosis is also increasing. However, as the amount of data that can be used with the next-generation sequencing technology increases, there is a growing need for techniques for accurately analyzing and processing large sequences of short sequences in a short period of time.

The most commonly used sequencing method for analyzing sequencing data is the variant calling analysis method, which has a problem in that it can not accurately diagnose gene deletion sites. Gene deletion is a mutation that occurs when a part of the chromosome is missing in the genetic recombination process and develops into a disease when genes that play an important role in gene expression are deleted. For example, the WAGR syndrome, which is characterized by unexplored development of the iris and various organs and mental retardation, is caused by the deletion of chromosome 11, the vocal crying sounds of cats, various physical and psychological abnormalities, It is caused by deletion. Therefore, although the deletion gene detection is very important for the diagnosis of the disease, the variant calling method has a limitation in detecting the deletion gene. US Patent No. 5830665 describes a technique for accurately diagnosing a gene deletion site, but it is not based on a next-generation sequencing method, and only a gene of a gene suspected to be deleted is subjected to nucleic acid amplification, As a comparison method, gene deletion is detected.

The present invention relates to a method for precisely distinguishing wild type, heterotypic deletion type and homo deletion type of a specific gene group by comparing results of lead distribution of next generation nucleotide sequence data. Korean Patent Laid-Open Publication No. 2014-0108177 discloses a method for determining the diagnosis of a gene abnormality using a read analysis. However, in view of analyzing the depth distribution and the GC content of read sequences, It differs from the invention.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and it is an object of the present invention to provide a novel method for sequencing a wild type, a hetero- To a method for detecting a deletion gene group based on the same.

However, the technical problem to be solved by the present invention is not limited to the above-mentioned problems, and other matters not mentioned can be clearly understood by those skilled in the art from the following description.

Hereinafter, various embodiments described herein will be described with reference to the drawings. In the following description, for purposes of complete understanding of the present invention, various specific details are set forth, such as specific forms, compositions and processes, and the like. However, certain embodiments may be practiced without one or more of these specific details, or with other known methods and forms. In other instances, well-known processes and techniques of manufacture are not described in any detail, in order not to unnecessarily obscure the present invention. Reference throughout this specification to "one embodiment" or "embodiment" means that a particular feature, form, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the invention. Accordingly, the appearances of the phrase " in one embodiment "or" an embodiment "in various places throughout this specification are not necessarily indicative of the same embodiment of the present invention. In addition, a particular feature, form, composition, or characteristic may be combined in any suitable manner in one or more embodiments.

Unless defined otherwise in the specification, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

In the embodiment of the present invention, the term " next generation nucleotide sequence analysis method " refers to a new concept of nucleotide sequence analysis technology capable of reading a large amount of nucleotide sequence to a sample to be analyzed within a short time and generating a large amount of nucleotide sequence data Sequencing techniques such as those using Roche / 454, Illumina / Solexa and SOLiD (Michael L. Metzker, Aplications of the next-generation sequencing, Sequencing technologies the next generation , Nature Reviews Genetics, Vol.11, pp31-46, January 2010). The next generation sequencing process can be divided into the following three steps.

(One) Capture of exome

Targeted sequencing can be sequenced by sequencing whole genomes or only exosomes using next-generation sequencing to find the causative genes of the disease. Sequencing only the exosome region is advantageous in terms of cost and efficiency. In addition, since changes in genes are frequently seen as direct diseases such as cancer, changes in the nucleotide sequence in the exosomal region may be effective in locating the causative genes. In order to sequence only exomes, you need a library that can capture exomes only. The most commonly used is, but not limited to, SureSelect Human All Exon Kits (http: // www.genomics.agilent.com). The SureSelect Human All Exon Kits were designed based on the exons of the human genome set by CCDS (Consensus CDS, NCBI, EBI, UCSC and Wellcome Trust Sanger Institute) and contain 1.22% of the human genome have.

(2) High-capacity parallel DNA sequencing

Next Generation Sequencing (NGS) can perform sequencing more quickly and at a higher rate than conventional capillary sequencing, The amplification process of the sample is omitted so that the experimental error caused in the process can be avoided. NGS systems manufactured by three companies are mainly used. Roche's 454 GS FLX, launched in 2004, is the first NGS instrument to introduce sequencing using pyrosequencing and emulsion-polymerase chain reactions. And the specific base can be identified according to the intensity of the light coming from the final stage of the experiment. When running for 7 hours, 100Mb sequence can be confirmed, and the existing ABI 3730 device shows much higher performance than 440kb sequence can be identified at the same time. Illumina Genome Analyzer from Illumina introduces the concept of sequencing by synthesis. After attaching a piece of DNA consisting of only one strand on a glass plate, the pieces are polymerized to form a cluster. . During this process, sequence identification is performed while confirming the type of base attached to the DNA fragment to be examined. In about 4 days of operation, 4-5 million fragments with a base length of 32-40 are produced. Sequencing by Oligo Ligation (SOLiD) from Life Technologies Inc. attaches a piece of DNA to a magnetic bead with a size of 1 μm and performs sequencing using an emulsifier-polymerase chain reaction. When sequencing is performed, a method of repeatedly attaching fragments of 8-mer is used. The base used for actual sequence identification is located at the 4th and 5th positions of the 8-mer. A fluorescent material is attached to the rest of the DNA to indicate which base is complementary to the DNA fragment to be examined. After 5 cycles of 8-mer every 5 cycles, 5 cycles of DNA sequencing can be performed. A feature of the SOLiD device is two-base encoding sequence identification, which identifies the same site in two sequence identifications when determining the sequence of a single base. Sequence identification is performed while moving the sequence one base at a time in one coupling cycle toward the adapter attached to the magnetic beads. This process has the advantage of eliminating the errors that occur in the sequence verification experiment.

(3) Analysis of nucleotide sequence data

In order to find the gene responsible for the disease, it is necessary to investigate what changes have occurred from the existing gene sequence, so that the sequence of the individual (patient) sequence reads is compared with the reference genome. This operation is called mapping. After finding the difference between individuals and reference sequences through mapping, it is possible to select a suitable selection criterion and extract only reliable base sequence mutation information (Variant Calling). This mutation information is either a single nucleotide variation (SNV) 4 or a short insert. Then, the nucleotide sequence variation information is compared with an existing database (dbSNP) to determine whether the mutation is a newly discovered mutation. And whether the mutation will result in a change in amino acid or not, and what effect it will have on the protein structure. This process is called annotation. Information on single nucleotide sequence mutations and short insertions / deletions extracted can be used to increase the quality of the information or to search for mutations in the cause of the disease through integration studies with the Genome Wild Association Study (GWAS) .

In one embodiment of the present invention, the " lead distribution diagram " is a distribution diagram of the leads of each gene constituting the target gene group in the gene group, and can be expressed in% as shown in Fig. 4 of the present invention.

In one embodiment of the present invention, the gene " wild type " refers to a gene of natural inheritance (normal state) without genetic abnormality, which inherits the inherent type of the gene possessed by wild type organisms in the genetic trait.

In one embodiment of the present invention, " gene deletion " is a mutation that occurs when a part of a chromosome is missing in a gene recombination process, and develops into a disease when a gene that plays an important role in gene expression is deleted. For example, the WAGR syndrome, which is characterized by unexplored development of the iris and various organs and mental retardation, is caused by the deletion of chromosome 11, the vocal crying sounds of cats, various physical and psychological abnormalities, It is caused by deletion. In addition, in the case of cancer development, it is observed that a gene abnormality occurs in a cancer-suppressing gene due to a gene deletion, and the regulatory function of the cell division is lost, thereby developing into cancer. Gene deletion can be divided into "heterozygous deletion" and "homozygous deletion". Dissociation deletion refers to the occurrence of gene deletion in only one of a pair of specific gene junctions, and homozygous deletion means that the same gene deletion occurs in both of a pair of specific gene junctions.

In one embodiment of the present invention, " BIM gene " is also referred to as BCL2L11 (Bcl-2-like protein 11) as a gene belonging to the BCL2 gene group. It is widely involved in various activities of the cell as a modulator of anti-apoptosis or pro-apatosis, together with other family genes belonging to the BCL2 gene family. Activation of the BIM gene increases cell death, and conversely, degradation of the BIM gene by its abnormality has the function of inhibiting cell death. Degradation of the BIM gene may increase the incidence of cancer by inhibiting the cell death of abnormal cells or mutant cells, which may be, but not limited to, lung cancer.

In one embodiment of the present invention, the term " diagnosis " means identifying a presence or characteristic of a pathologic condition. For the purpose of the present invention, the diagnosis includes identifying a genetic defect group causing congenital genetic diseases or cancer, , But is not limited to.

In one embodiment of the present invention, the term " screening " is to select a substance having a specific property aimed at from a candidate group made of various substances by a specific manipulation or evaluation method. For the purpose of the present invention, the screening of the present invention may refer to a series of processes for identifying a genetic defect group causing congenital genetic diseases or cancer, etc., Can be used to measure changes in protein activity.

In one embodiment of the present invention, " neovascularization " refers to a cell phenomenon in which vascular endothelial cells are proliferated and reconstituted to form new blood vessels from an existing vascular network. In order for cancer cells to proliferate and grow, new blood vessels are needed to supply oxygen and nutrients, thus inhibiting cancer metastasis through inhibition of neovascularization.

In one embodiment of the present invention, " metastasis " refers to the cancer cells leaving the primary organs to another organ. The spread of cancer to other parts of the body is largely divided into the development of cancerous tissue in primary cancer, direct invasion of the surrounding organs, and distant metastasis along the blood vessels or lymph vessels to other organs far away. Metastasis can be regulated by inhibiting the expression of a gene associated with cancer development or inhibiting the protein activity of the gene.

In one embodiment of the invention, analyzing the sequence of the sample; Comparing and analyzing the desired gene sequence with the reference sequence; And analyzing a lead distribution degree of a target gene group. When a value less than 50% of the highest value appears in the lead distribution map, the method provides a method of discriminating the gene as a deletion deletion gene, A method for distinguishing a heterozygous deletion gene using a next-generation sequencing method in a step of analyzing a sequence of a sample, wherein the heterozygous deletion gene comprises a heterozygous deletion gene having a deletion gene size of 20 to 3000 bp Wherein the heterozygous deletion gene provides a method for identifying a heterozygous deletion gene that is a BIM, and the heterozygous deletion gene provides a method for identifying a heterozygous deletion gene that is a lung cancer inducing gene.

In another embodiment of the present invention, there is provided a method of analyzing a sample comprising: analyzing a sequence of a sample; Comparing and analyzing the desired gene sequence with the reference sequence; And comparing the number of desired gene groups (X) with the number of lead distribution groups (Y), and when XY is greater than 0, determining the number of homozygous deletion genes as the number of homozygous deletion genes, A method of identifying a deletion gene provides a method of identifying a homozygous deletion gene using a next-generation sequencing method in a step of analyzing a sequence of a sample, wherein the homozygous deletion gene is a homozygous deletion gene having a deletion gene size of 20 to 3000 bp Wherein said homozygous deletion gene provides a method for identifying a homozygous deletion gene that is a BIM, and said homozygous deletion gene provides a method for identifying a homozygous deletion gene that is a lung cancer inducing gene.

In another embodiment of the present invention, there is provided a method for analyzing a sample, comprising: a sequence analyzer for analyzing a sequence of a sample; A comparative analysis unit for comparing and analyzing a target gene sequence and a reference sequence; A lead analysis unit for analyzing a lead distribution of a target gene group; And a discriminator for discriminating the gene as a deletion deletion gene when a value less than 50% of the maximum value is found in the lead distribution map, wherein the heterozygous deletion gene discrimination apparatus comprises a sequence- A method for identifying a heterologous deletion gene characterized by using a sequence analysis method.

In another embodiment of the present invention, there is provided a method for analyzing a sample, comprising: a sequence analyzer for analyzing a sequence of a sample; A comparative analysis unit for comparing and analyzing a target gene sequence and a reference sequence; A lead analysis unit for comparing the number (X) of desired gene groups and the number (Y) of lead distribution groups; And a discriminator which discriminates the number of homozygous deletion genes when XY is larger than 0, and wherein the homozygous deletion gene discrimination apparatus uses a next-generation sequencing method in the sequence analysis unit And a homologous deletion gene.

Hereinafter, the present invention will be described in detail.

Since gene deletion develops into disease, it is very important to identify gene deletion sites in sequence analysis. Unlike the conventional variant calling method, the nucleotide sequence analysis method of the present invention can distinguish the wild type, the heterotypic deletion type and the homo deletion type of a gene accurately, And monitoring is expected to be greatly utilized.

FIG. 1 is a diagram showing the results of managing the quality of a library for next generation sequencing analysis (NGS) according to an embodiment of the present invention.
FIG. 2 is a diagram showing a result of variant calling analysis of nucleotide sequence variation of a heterozygous deletion BIM gene sample (18-BIM-Hetero) according to an embodiment of the present invention.
FIG. 3 is a diagram showing an analysis result of sequencing data of the BIM gene directly confirmed by IGV (Integrative Genomics Viewer).
FIG. 4 is a bar code summary result showing the result of analyzing the read coverage of the position of the BIM gene.
FIG. 5 is a view showing the results of read coverage analysis of the position of the BIM gene. FIG.

Hereinafter, the present invention will be described in more detail with reference to Examples. It will be apparent to those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example 1: Next Generation Sequence Analysis (NGS)

Example 1-1. DNA extraction from paraffin block samples

In lung cancer patients, lung and lymph node tissues, which were separated and discarded during surgery, were kept in a paraffin block state and DNA (gDNA) was extracted from lung and lymph node tissue paraffin blocks using QIAamp DNA extraction kit (Qiagen, Germany). The details are as follows. For the isolation of gDNA, cancer tissues were cut from the tissues stored in a paraffin block state, and 1 ml of xylene was added thereto in a 1.5 ml microcentrifuge tube to dissolve the paraffin bound to the tissue. After centrifugation for 2 minutes, the supernatant was removed, 1 ml of ethanol was added to wash the tissues, and centrifugation was performed for 2 minutes. The supernatant was removed, and the mixture was allowed to stand at room temperature for 10 minutes to evaporate the alcohol. 20 [mu] l of protease K and 180 [mu] l of buffer solution were mixed and reacted at 56 [deg.] C for 1 hour. The reaction solution thus obtained was purified using a QIAamp spin column and washed twice with a buffer solution. Then, 50 μl of the buffer solution preheated at 70 ° C was dissolved to extract gDNA, which was stored at 20 ° C. For each extracted gDNA, the amount of gDNA was measured using a Qubit dsDNA HS Assay Kit and a Qubit 2.0 fluorometer (Flourometer; Life Technologies, USA).

Examples 1-2. Generation of library for next-generation sequencing (NGS)

Using 10 ng of gDNA stored at -20 ° C, a library for performing NGS was constructed using panel primers of target genes including 17 lung cancer-related genes. The panel primers are shown in Table 1.

Forward primer Reverse Primer Target gene TCCTCATGTACTGGTCCCTCAT GGTGCACTGTAATAATCCAGACTGT KRAS CATACGCAGCCTGTACCCA GTGGATGCAGAAGGCAGACAG RET TGTCCTCTTCTCCTTCATCGTCT AGGAGTAGCTGACCGGGAA RET CCATCTCCTCAGCTGAGATGAC GGACCCTCACCAGGATCTTG RET CCTATTATGACTTGTCACAATGTCACCA TAGACGGGACTCGAGTGATGATT BRAF CACAGCAGGGTCTTCTCTGTTT CCTTCTGCATGGTATTCTTTCTCTTCC EGFR TGTCAAGATCACAGATTTTGGGCT ATGTGTTAAACAATACAGCTAGTGGGAA EGFR GGTGACCCTTGTCTCTGTGTTC AGGGACCTTACCTTATACACCGT EGFR GGAAACTGAATTCAAAAAGATCAAAGTGCT GGAAATATACAGCTTGCAAGGACTCT EGFR cttACCAGCTTGTTCATGTCTGGA AGAGGACTTCGCTGAATTGACC MET GCAGCGCGTTGACTTATTCATG CACAGCTACTCTCAGAAAGCACT MET AACTGAGCTTGTTGGAATAAGGATGTTAT CCATTTTGGTTTAATGTATGCTCCACAATC MET CCCATCCAGTGTCTCCAGAAGT CAAGTGACACTGGTTGTAAATATGCATTT MET GTTATGACAGGATTTGCACACATAGTT CCCAAGCCATTCAATGGGATCT MET CCCTTCTCTTCACAGATCACGA CAGACAGATCTGTTGAGTCCATGT MET GCTTGGGCTGCAGACATTTC GCCAGCTGTTAGAGATTCCTACC MET TGGTCCTGCACCAGTAATATGC ATTATAAGGCCTGCTGAAAATGACTGA KRAS CCATCCACAAAATGGATCCAGACA GCTCTGATAGGAAAATGAGATCTACTGTTT BRAF AGGAAATTCCCACTTAGGAACCATTG AGCAAACTCAGTTGAAATGGTTTGG MET GTTCAGTGTGTCAAACAGTATTCTTGAATG GTTGGATGAATTTCATAGACAATGGGATC MET ACAAGCATCTTCAGTTACCGTGAA AAAACTGCAATTCCTCTTGACTATTCTACA MET GCTATGGATGTTGCCAAGCTGT TAACATGAAAAAGGCTTTGGTTTTCAGG MET GCAACAGCTGAATCTGCAACTC ATTTTCATTGCCCATTGAGATCATCAC MET CTGTGTTTAAGCCTTTTGAAAAGCCA CCAAGTACAACAATTGTATTCACATAGCT MET CATAATTAAATGTTACGCAGTGCTAACCA GCAAACCACAAAAGTATACTCCATGGT MET CAGTCAAACCCTCAGGACAAGA CCCTCGGTCAGAAATTGGGAAA MET TCTCAGGAATCACTGACATAGGAGAAG CGAATGAAATTTCGAAGATCTCCATGTTT MET GCTGGTGGTCCTACCATACATG TTTTTAAAGACTCAGAGCAGGCCTATT MET GAGGCCAGATGAAATACTTCCTTCA AAAATCAGCAACCTTGACTGTGAATTTT MET TGTCCTTTCTGTAGGCTGGATGA GGTGGTAAACTTTTGAGTTTGCAGA MET GTGAAGTGGATGGCTTTGGAAAG AAACTGGAATTGGTGGTGTTGAATTTTT MET CGTCTCCTGGAGATGGATACTCT CTGTGGAGGAACTTTTCAAGCTG FGFR1 GCAGTTACTGGGCTTGTCCAT GAGGCGGAGAAGCTCTAACAC FGFR1 GCATGGACAGGTCCAGGTAC GGAAGACCTGGACCGCATC FGFR1 CCTTACCTGGTTGGAGGTCAA GGAGACGTCCCTGACCTTACA FGFR1 GAGTTCTTTGCTCCACTTGGGA CCACACTCTGCACCGCTAG FGFR1 GCACCTTACCTTGTTCAGGCAA TGGAGTATCCATGGAGATGTGGA FGFR1 AGGAATGCCTTCAAAAAGTTGGGA TCCAGTGCATCCATGAACTCTG FGFR1 GTGATGGCCGAACCAGAAGAAC CATGTGCCTCTGCCATTGTTG FGFR1 GAGAGAGGCCTTGGGACTGATA TCAGAAATGGAGATGATGAAGATGATCG FGFR1 AGCAGGTTGATGATATTCTTATGCTTCC CCACTCCCTTAGCCTTTATCCTG FGFR1 TTAAACCCAATGCCCAGACCCAAA CCCTTCTTCTTCCCATAGATGCTCT FGFR1 TCTCCTCTGAAGAGGAGtcatcatc GGTGTCCGTGTTCATCTGGAAC FGFR1 GGCAGAAAGAGGACTCCTCAGT CGACTGCCTGTGAAGTGGATG FGFR1 TGTAGATCCGGTCAAATAATGCCTC GGTTTCATCTGAGAAGCAAGGAGT FGFR1 GCATTAGAGGCCCAGAGAGAGA TCATCGTCTACAAGATGAAGAGTGGTA FGFR1 GCTGTGGAAGTCACTCTTCTTGG CTAACACCCTGTTCGCACTGA FGFR1 TTGGAATGGGACAAGATTTTCTTTGC CGAAAGACTGGTCTTAGGCAAAC FGFR1 AGGACAGAAGCATCACTTACACTTC CAACTTATGCCACTCTCTGTTTCC FGFR1 AAATGAAAAGCATGTAATCAGGACTTCCTA ACACTGCGCTGGTTGAAAAATG FGFR1 TGTGGTCAGGTTTGAATTCTTTGC GCCGTAGCTCCATATTGGACATC FGFR1 CATGCAATTTCTTTTCCATCTTTTCTGG CTCACAGGTGTTGGGCAGAT FGFR1 CTTTGTCATTTACAAGTACTTTGCAAACAC TCCCTAAAGCTGGAGTCCCAAATA ROS1 TTCTAGTAATTTGGGAATGCCTGGTT cgcctcTGAATATTTCTTTAATGTTGTCTT ROS1 GCCTAGGTGCTCCATAATGATGG AGTCTGGCATAGAAGATTAAAGAATCAAAA ROS1 ACCAATCATGATGCCGGAGAAAG ACCTGGTGTGGTTGTCAATACC ALK TCACCGAATGAGGGTGATGTTTT GCAGAGCCCTGAGTACAAGC ALK GGTTGTAGTCGGTCATGATGGT TGGCCTGTGTAGTGCTTCAAG ALK ccacttcacctagccAGAATTTTC CTGACAGGCGATCTTGAACATCA EML4 AGATGATAGTATTTCTGCTGCAAGTACTTC CACATGATCTTCAGAGATTGCAAGAC EML4 CAAGAAGATGAAATCACTGTGCTAAAGG CAGCTTCAACTTTCAAAGAAAATATTGCAA EML4 CTCTGTCGGTCCGCTGAAT GCTGTGTGCGGAAGGAAAAAA EML4 GTTCTAGTTCAGTCTTCTTCATTGTACCTT CGGAAGAAGGTAAACATTAGCTCTACAG EML4 TCTGTTAAGATATGGTTATCGAGGAAAGGA CAACCATTTCACACAGTCTGTATGG EML4 AGAGAACTCAGCGACACTACCT TCTGAGCTTTCCACAAGAAATCACTT EML4 GCTTCCAAATAGAAGTACAGGTAAGCT CTGAGCACATGTCAAGAGCAAATC EML4 TCTTGCCACACATCCCTTCAAA GCCAGTGTGAGGAGTTTCTGTG EML4 ATGGTATTTCTTTCTTAGAATGTTAGCCCTATC AAATTTACCTTTATTTCTGCTCCTTTTGCTTT EML4 GAGCATATGCTTACTGTATGGGACT GCTTTTGCGACTTACGAATAGATAGTTCTA EML4 TTTTTCATTTGTTTAAATGTGTATTGTTCCATG GGAGAAGGTGATGCTCGAATTTG EML4 gaggaaTCTCATTCTAATGATCAAAGTCCA CCATTCCCTAGCTCTGTACTTGG EML4 TGTCAGTTACATGTCTTTGATACTCAGAA GGGTGTTGAAGGTATTTTCGACAATTTAT EML4 CTTGGGAAAATTCAGATGATAGCCGTA CCAGACATACATAACTGTACATGCAAACAT EML4 CCATAGGGAGACTTTCTCATGTACTC TAGCATTTAAACATCCCACCACTTCA EML4 CCCTCCTTCCAAATGGACTTAATTTTAAA TGCACCACTTCCATTGGTTATACAG EML4 CCTCGAGCAGTTATTCCCATGTC AGACAATTCTGCAATGTTAGTTTTTCCC EML4 aacttttacagttttcttgaggtgattttaatgg TTTCAATTAGCAAAAATTAAACTAAGAAGGGCA EML4 ACAAAGGTATTCTGTTGTTTCATGTTTCC CAATGTCTCTAGGTACAGAAGGCAT EML4 GTTCTACTGAAGTTTTCTTCCAAATAGACACT CTCCAGTTAATAACATCCCATTTCTCATCT EML4 GGCAGTGTGTTCACACTTTGTC GTGGGACAAAATACCTGAGTTTAGAGTATT EML4 GACTAAAACTTTGAAGTAGTCATTTTTGTCTT GAATGAACATGGTAATTGGCCGA EML4 TGTCTTGTGTTTCAACAGAAGGAGAATAT GCTGTCATGGTAGAGAAGGATACG EML4 CCTGAGAAGCTCAAACTGGAGT cctggccTGATGTTTCCTTTTTAATTTTT EML4 gcacaccagcgttatgacaaag TGTGTGGATAGAAACTAGATCTCTGGTT EML4 GGTGGTTTGTTCTGGATGCAGA CAATTGCAGTGAAGTGCTGTGAAT EML4 AGTGGATAGGATTCATTCATTAATTGCCA TACAGCCAGGAAGGTACCATCT EML4 ATTTCTCTAGTCAACACTGACCTATTTTATTCT TCCCAATAATTTTACAACTTGTTCTACTTCACT EML4 GTAGCAGTAATTGAATTGATACTTGAAGGAGA CGCTCCAGGTCCAGAAGAAAATATG EML4 GCAAATACCATAATTACATGCGGTAAATCT GCCATGACAACTTGATGCTTATTAAACAAT EML4 TTTTTAAATGGCATTAGTTCTGTGTGCT GCTCAAAAGTGCCAAGTCCAATA EML4 TCTGTTACTCTATCCACACTGCAGAT ACTTCATGGCCACATAAACACAAAAC EML4 AACAGTATTGGCTAGCTGTTGAACT ATACTTACAGTACAATATTTCATAGTCTCCCGA EML4 CCCAGACAACAAGTATATAATGTCTAACTC CCCTGACAGACACATCTTAGCatatatata EML4 CAAGCACTATGATTATACTTCCTGTTTCT ACAAACCACTTCTTTACATCAGGTGT EML4 TTAATAAGCATCAAGTTGTCATGGCAAAAAA GGCTCTACAGTAGTTTTGCTCCATA EML4 AGACTCAGGTGGAGTCATGCTTA CCTGGTCTAAGAGATGGGACTGA EML4 ATTTCTGAAACAGGCATGTCAAGAATG CCAGTTGATATCAGGTGACTGTCATTG EML4 AGCCATGTCACCAATGTCAGTTTTA GGCTTTGGTTAGAGTAGTATCCGCTA EML4 AGTTATCTTTGCCTCAGAATGAGACTG GTGGGAGAACTGCTTATTCTACTTTCC EML4 GCCCTTAAATGAGACAGCTGAAGA GCTTATCTCGTTGCATGGCTCTT EML4 GAGACCTTGGTGAGCCTCTTTATG ACATGCAGCTGAAGGAAAAGAGTT EML4 CAGCTATAAATGCAGGCTTCGAGTA GTTCCTCGTAAAATAAAGTTTCGTGATGT EML4 GGAAAGGCAGATCAATTTTTAGTAGGC ACCCTGAAAATGAAAGACACTCATTGTTAT EML4 GCTAATTTTTCTGCATCCCTGTGTT GGGATACTGAAACAGATGGACTTTACAAA EML4 GTGATAGCTGTTGCCGATGACT GAGTATCATGGAGAGGAATCAGTAACCTAT EML4 CTTTTGATGACATTGCATACATTCGAAAGA CAGTTATCTTTTCAGTTCAATGCATGCT PIK3CA ATCCGCAAATGACTTGCTATTATTGATG CCCAGGATTCTTACAGAAAACAAGTG NRAS GGGTGAGGCAGTCTTTACTCAC GCCGTTGTACACTCATCTTCCTAG ALK CCTCACCTCTATGGTGGGATCA GCTCGCCAATTAACCCTGATTACT NRAS CAGGTCTCTCCGGAGCAAA GCCAAGTCCCTGTGTACGA HER2 AGAACCTCTCAACATTGTCAGTTTTCT GCTCTGAGTAGAACCATTGCTCA MET TTGGCACAACAACTGCAGCAAA CCAGAACATTGTTCGCTGCATTG ALK GTCTCTCGGAGGAAGGACTTGA CAGACTCAGCTCAGTTAATTTTGGTTAC ALK CAGCTGGTGACACAGCTTATG CTCCGGAGAGACCTGCAAAGAG HER2 TATGCAGATTGCCAAGGTATGCA AATGGGAAGCACCCATGTAGAC HER2 GGGTGTCTCTCTGTGGCTTTAC ACTCTGTAGGCTGCAGTTCTCA ALK GCCAATGAAGGTGCCATCATTC CTCAGGCATCCCAGGCACAT AKT1 ATTTTACAGAGTAACAGACTAGCTAGAGACA AAAGAAAAAGAAACAGAGAATCTCCATTTTAGC PIK3CA GAAATTTAACAGGGTGTTGTTGTGCA CTGTTCATCTGACAGCTGGGAAT DDR2 GCTGGAGGAGCTAGAGCTTGAT GCTTGTGGGAGACCTTGAACAC MEK1 TGGGTGGTCAGCTGCAAC CATGCTTCAATTAAAGACACACCTTCTTTAA ALK GCTCTGAACCTTTCCATCATACTTAGAAAT ccagactaacaTGACTCTGCCCTATATAAT ALK AAAGAAGGTGTGTCTTTAATTGAAGCAT gggtctaatcccatctccagtct ALK TCAtgttagtctggttcctccaaga gggttatacttgcaacacagtct ALK agggaaggctgggtgaacc actgactttggctccagaacc ALK ggagcctaaggaagtttcagcaag cactgctgtgattgcactgaag ALK ggttctggagccaaagtcagtc aactataggaaacacaactgaccaagatc ALK caatcacagcagtggatttgagg aggcggaattagagcacagatc ALK GAAGAGCCACATCAtgaaaagatctct agttaccatccctgcctacaga ALK ggacctctttggactgcagttt ggtagagctctttaggatttttcaaaacca ALK ggttgtcaatgaaatgaattcaccaacata ACAGAATCTACCCACTGAATCACAATTT ALK AAACTCCATGGAAGCCAGAACA ttcattcgatcctcaggtaacccta ALK tggaccgaccgtgatcagat ATCTGCCGGTAGAAGGGAGAT ALK CCTTTGAGGGATGGCACCATAT GAGACATGCCCAGGACAGATG ALK CCTTTCCCTCTGCCCTTTTCAA AGAGAGATAGGAAAATCGGTTTCTGAGTAT ALK GGCTCACAGGCTGAACAGAAAT ACTTCTAGCTCCCACATGCTTC ALK CATTACATAGGGTGGGAGCCAAA TGTGTATCCTCCTGGCTGATCA ALK GCTTTCACCATCGTGATGGACA AAACGGAAGCTCCCAACCTT ALK CTGATCAGCCAGGAGGATACAC CCAAGGTGTCACTTCGTTATGC ALK CCCACCCAATTCCAGGGACTA GGCTTTCTCCGGCATCATGAT ALK TGCTTTTCTAACTCTCTTTGACTGCA GATTGTGGCACAGAGATTCTGATACTT MET CACAGCCTGAGACACTATTCAGTC ACTCTCGCTGATCCTCTCTGT ALK ATGTATTTAACCATGCAGATCCTCAGTTT ATCTTGTTCTGTTTGTGGAAGAACTCT PTEN GGATGAGCTACCTGGAGGATGT CCATCTGCATGGTACTCTGTCT HER2 TCTTTGTCTTCGTTTATAAGCACTGTCA GTGTTCTTGCATAAAAACACTTCAAATG ROS1 TGAAACTTGTTTCTGGTATCCAAAAATCAT CTGGCTTGCAAAAATCCAGTAGTAG ROS1 TTCCTTTAGGAAATGTTAACAGTGCATTTG GATTAAAAATGGTGTAGTATGATTTGTGTACTT ROS1 ACTTACCAAAGGTCAGTGGGATTG CTCTGTGTGCTTAGGTAGAGCTG ROS1 CTGTGACCACACCTGTCATGTA AAGAAGGCAAGACCCTTAAAGGAG RET AGCTCTACCTAAGCACACAGAGTAATA GTGGTTTGTTgctctctgcaaaaa ROS1 GGCCCAATGTGTGGATAGAAC CAGGACAACTTCTCTACATAGCCA RET GTGTGGATAGAACTTTGGTGGGA GAAGTGTCCAGGACAACTTCTCT RET GGGTGGCTATGTAGAGAAGTTGT CTACATGACAGGTGTGGTCACA RET CGAAGTACTGAGTCCAAGCCAT GACAAGTTCCAATGTGCAGAGAAC RET CTTCTGCACTGAAATCTTTCTACAGAATATATT gaatctagataccttgccggtgaag ROS1 cgactagaagcaactccgttca gctcactgctcttccttctctct ROS1 tagattcgctcatcaaaattgactagaagg gcacagttctttggaaaagcaatttc ROS1 gggaaattgcttttccaaagaactg cccagggagttcagtaagcttag ROS1 atcaaaagcaaggtgtttttgcttt ATAATGCCAACTATTTAGTATCCAAAGACTGAG ROS1 AAAAACTAATTAAATCCAGGTAAAAAGCCAT TCGTTTATGGGTGATTTTGACAAATAAGTTTT ROS1 GCCATATATAAGTACACAAATCATACTACACCA GTTTGTTTTGGTTTATTTTGACTCGTTTATGG ROS1 TCACCCATAAACGAGTCAAAATAAACCA CAATGATAAACACTCTTGTACTCTGCaaaa ROS1 CTGCACATTGGAACTTGTCCATG TTCTCCTAGAGTTTTTCCAAGAACCAAG RET GTGGGAATTCCCTCGGAAGAA TGCCTGGCAGGTACCTTTC RET AGTCACTGTCCCTGTGACCAT ACGTAGGGCTATATAATACCAGAAAACTCA RET GCATAGGGACACGTTTCTGTCAT AGGCTGTGCTCATTACACCAG RET CATTTGGAACAGAGGAAAATTTTGACCTC AGaccactattgccctcttacaga RET CCAGTGCCAGCTGGTGTAA GTGGGAAGGCCTGAGAACAC RET GGGCAGTAAATGGCAGTACC TCGGCACCACTGGGTACAG RET TCACATCCAGGTTATATTCCTCAGTAGAAA gtaagcttcgttccaatactttttctacat ROS1 TTTAGGACCAAGAAATCTCAGTCTTTGG GTTTCCTCTACACAACTGAAACTACCT ROS1 CTCAGTCTTTGGATACTAAATAGTTGGCA tcctcatgccatagtttgccag ROS1 GTCCCAACCATGTCAAAATTACAGAC CCATGGGCTAGACACCACT HER2 atttgctcttccatgacaggctta caagtatctcctgatggatgaatgga BIM ccagtgtgtgtatatcacatacttcattta agcaattccacttccaagtatatatccaaaaa BIM AAAGTTAATGTACCGAGGTAAGTTTTCAGT CTGTAGAATGTCCAGAGAAAATTATGGACT BIM GAAAGGACTTAGCCAGATGTGAGTTT CAAAGTCAAGAGAACCACTTATCAACTCA BIM GTCTTAGTTCATGCCTGAAGACCA TGACTTCTTTGTGGAAAATGTATTTTGCAA BIM ATTCTTTACTCAACCCTATCCATGAAGTTC CTGGCTAAGGCGAACCTCTTTAT BIM CATTTCTAAATACCATCCAGCTCTGTCT CATGTGTAGCTGCTGGGATG BIM GCACACCTGTGAGGTGGTG CTGAAATGAGTTCACGAGCAGTAGTA BIM GGGCTGGAAGTTTTATTATTGCTGT CCTGTTAACTCATTTAGTAAGCAAGGATGT BIM GTTAACGTCTTCCTTCTCTCTCTGT TGAGGTTCAGAGCCATGGAC EGFR CATGCGAAGCCACACTGACGT CTTTGTGTTCCCGGACATAGTCCA EGFR TCATCACGCAGCTCATGCCCTT GTGAGGATCCTGGCTCCTTATCTC EGFR gctggtACTTTGAGCCTTCACAG CACCAGCCATCACGTATGCTTC HER2 TCTCCCATACCCTCTCAGCGTA CAGCCATAGGGCATAAGCTGTG HER2

Library production for NGS was performed using the Ion AmpliSeq library kit 2.0 (Life technologies, USA). First, DNA primers were amplified from the DNA extracted from the sample using a panel primer pool which was preceded to obtain only the mutation site of the target gene. 4 × 5 × HiFi master mix, 10 μl of panel primer pool, and 10 μg of DNA were mixed and adjusted with sterilized distilled water to make the total reaction volume 20 μl. The prepared mixed reaction solution was amplified by repeating 21 cycles of 2 minutes at 99 ° C, 15 seconds at 99 ° C, and 4 minutes at 60 ° C in a PCR instrument. The resulting amplification product was subjected to a reaction at 50 ° C for 10 minutes, 55 ° C for 10 minutes, and 60 ° C for 20 minutes, and both ends of the amplification product were partially cleaved by adding 2 μl of Fupa solution. 2 μl of Ion P1 adapter and 2 μl of Ion Xpress barcode were added to the partially digested amplified product, and the reaction was performed at 22 ° C for 30 minutes and at 72 ° C for 10 minutes to bind the sequencing adapter and the sample to the amplification product . The amplification product, in which the sequencing adapter and barcode were combined, was washed with 45 μl of AMPure XP solution.

Examples 1-3. Library quality control for next-generation sequencing (NGS) (Q.C)

The length and the amount of the library constructed using the High Sensitivity chip were measured using an Agilent Bioanalyzer 2100 (Agilent, USA) in order to check whether the prepared NGS library could be used for sequencing. The length was 100 to 300 bp, A library satisfying the condition of 100 pmol / l or more was used for sequencing. The results of library quality control using the High Sensitivity chip are shown in FIG.

Examples 1-4. Sequencing of Next Generation Sequencing (NGS)

As shown above, the emulsion PCR (emPCR) for 8 pM of the library, which is composed of the amplified products integrated for each sample and to which the bar code sequence of the sample was attached, was applied to the Ion PGM Template OT2 400 Kit Catalog No. 4479878) and the corresponding manual (Ion PGM Template OT2 200 Kit, Publication Number MAN 0007218, Revision 3.0). Template-positive ISP (Ion Sphere Particles) were reacted at 50 ° C for 2 minutes, and then ISP was incubated with DynaBeads MyOne Streptavidin C1 Bead (Life Technologies, USA) with Enrichment ). Personal Genome Analyzer (PGM) sequencing was performed using the Ion 318 chip kit v2 (Life Technologies, USA; Catalog Number 4484354) and the Ion PGM Sequencing 200 kit (Life Technologies, USA; Catalog Number 4482002) Publication Number MAN0007242, Revision 2.0). Meanwhile, the PGM (Personal Genome Analyzer) sequencing technique using the Ion PGM sequencing 200 kit and the Ion 318 chip kit can directly capture the change in pH, which occurs when DNA synthesis takes place in a small hole of a semiconductor chip, It is a very fast sequencing platform that can be decoded within 4 hours by principle. In addition, the PGM (Personal Genome Analyzer) sequencing technology has a multiplexing sequencing capability capable of simultaneously reacting various samples.

Example 2: Software configuration for analysis of lead distribution for target gene mutation

Sequence data of short amplification products obtained by PGM (Personal Genome Analyzer) sequencing was analyzed using Ion Torrent Suite 4.0.2 software. Ion Torrent Suite 4.0.2 software was used to sort NCBI's hg19 reference sequence and the results of the sequencing data for each sample were uploaded to Ion Reporter's cloud system for mutation analysis. In order to effectively analyze the target gene included in the panel developed in the present invention, the analysis time is applied by applying the BED file so that only genes included in the panel can be analyzed, and the data quality stringency value Were used for analysis. In order to detect somatic mutations with low frequency, more than 3 sequencing leads were designed to perform the analysis. In order to increase the accuracy of mutation detection, mutations detected in more than 10 sequencing leads were shown. The information of the detected mutations was derived from mutation information linked with databases of highly reliable COSMIC, dbSNP, ClinVar, VariantDB, Variant KB, 5000Exomes, OMIM and DrugBank. In addition, in case of BIM gene, about 2,000 bp deletion mutation occurs. Since mutation detection is not easy analytically when a deletion site is long, 9 amplification products designed in BIM gene are separated The presence of long-deletion-induced specimens and the long deletion mutations were confirmed by homology or heterozygosity analysis through coverage analysis.

Example 3: Analysis of lead distribution for target gene mutation

As a result of the analysis by variant calling of only the nucleotide sequence variation information, 7 out of 10 samples of the BIM gene among the panel primers were deleted samples (six homologous deletion gene samples and one homozygous deletion gene sample) Detection of gene deletion was not made, though. This means that the Variant Calling method is insufficient to detect deletion genes important for disease diagnosis. Fig. 2 shows the result of the variant calling analysis of the nucleotide sequence of the heterozygous deletion gene sample or the BIM gene sample (18-BIM-Hetero) without deletion detection.

Secondly, sequencing data of the BIM gene was confirmed directly by IGV (Integrative Genomics Viewer). As a result, there was no lead in homozygous deletion samples, but it was impossible to distinguish between wild type (WT) and heterozygous deletion genes. The results are shown in Fig.

Finally, we analyzed the read coverage of the BIM gene by applying the BED file to analyze only the genes contained in the panel. As a result, the distribution of wild type gene was more than 3%, while that of heterozygous deletion gene was less than 3% and that of homozygous deletion gene was 0%. This means that it is possible to distinguish between wild type, heterozygous deletion and homozygous deletion genes as a result of analysis of lead distribution of a specific gene group. The results are shown in Fig. 4 and Fig.

Claims (14)

Analyzing the sequence of the sample;
Comparing and analyzing the desired gene sequence with the reference sequence; And
And analyzing the lead distribution of the target gene group,
If less than 50% of the maximum value is found in the lead distribution map, it is determined as a heterozygous deletion gene.
The method according to claim 1,
The method for discriminating the heterozygous deletion gene uses a next-generation sequencing method in the step of analyzing the sequence of the sample.
The method according to claim 1,
Wherein said heterozygous deletion gene is a heterozygous deletion gene having a deletion gene size of 20 to 3000 bp.
The method according to claim 1,
Wherein said heterozygous deletion gene is a BIM.
The method according to claim 1,
Wherein the heterozygous deletion gene is a lung cancer-inducing gene.
Analyzing the sequence of the sample;
Comparing and analyzing the desired gene sequence with the reference sequence; And
(X) of the target gene group and the number (Y) of the group of lead distribution groups,
When XY is greater than 0, it is determined as the number of homozygous deletion genes.
The method according to claim 6,
The method for discriminating the homozygous deletion gene uses a next-generation sequencing method in the step of analyzing the sequence of the sample.
The method according to claim 6,
Wherein said homozygous deletion gene is a homozygous deletion gene having a deletion gene size of 20 to 3000 bp.
The method according to claim 6,
Wherein said homozygous deletion gene is a BIM-homozygous deletion gene.
The method according to claim 6,
Wherein said homozygous deletion gene is a homozygous deletion gene.
A sequence analyzer for analyzing the sequence of the sample;
A comparative analysis unit for comparing and analyzing a target gene sequence and a reference sequence;
A lead analysis unit for analyzing a lead distribution of a target gene group; And
And a discriminator for discriminating the gene as a deletion deletion gene when a value less than 50% of the maximum value appears in the lead distribution map.
12. The method of claim 11,
Wherein the heterozygous deletion gene discrimination apparatus uses a next-generation sequencing method in a sequence analysis unit.
A sequence analyzer for analyzing the sequence of the sample;
A comparative analysis unit for comparing and analyzing a desired gene sequence and a reference sequence;
A lead analysis unit for comparing the number (X) of desired gene groups and the number (Y) of lead distribution groups; And
And a discriminator for discriminating, when XY is greater than 0, the number of homozygous deletion genes.
14. The method of claim 13,
Wherein said homozygous deletion gene discrimination apparatus uses a next-generation sequencing method in a sequence analysis unit.

Images