KR101882866B1 - Method for analyzing cross-contamination of samples and apparatus using the same method - Google Patents

Abstract

Obtaining first sequence information of the nucleic acid fragment from each of the target sample and the additional sample, and second sequence information of the nucleic acid fragment from the mixed sample in which the target sample and the additional sample are mixed; Calculating allelic frequency from each of the obtained first sequence information and second sequence information; And comparing the calculated allele frequency with a specific locus of the chromosome, the method comprising the steps of: comparing the frequency of alleles of the sample with respect to the target sample; The method and apparatus can measure the degree of cross-contamination between samples at a particular chromosomal location, thereby imparting reliability to the mutation extraction results.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method and apparatus for analyzing cross-

To a method and apparatus for analyzing the degree of contamination between samples.

A genome is any genetic information that a creature has. Techniques for sequencing one individual's genome include DNA chip, next generation sequencing technology, and next generation sequencing technology. Next-generation sequencing can be used interchangeably with large-scale parallel sequencing or second-generation sequencing.

Analysis of genetic information such as nucleotide sequence and protein is widely used to find genes expressing diseases such as diabetes and cancer or to correlate genetic diversity and expression characteristics of individuals. In particular, genetic data collected from individuals is important in identifying genetic characteristics of individuals with different symptoms or progression of disease. Thus, genetic data such as individual nucleotide sequences, proteins, and the like are key data that can provide information on current and future disease-related information to prevent disease or to select optimal treatment methods in the early stages of the disease. Techniques for precisely analyzing and diagnosing mutations such as SNV (Single Nucleotide Variant), CNV (Copy Number Variation), InDel (Insertion and Deletion) and translocation related to diseases using these genetic information of living organisms are under study.

In the past, most of the cases of mutation detection were estimated using the general population frequency provided by a known database or not considering the influence of contamination between samples. However, there is a need for techniques for measuring or correcting the effects of interferences between samples in order to detect mutations having a low allele frequency.

According to an aspect, there is provided a method for detecting a nucleic acid fragment, comprising: obtaining first sequence information of a nucleic acid fragment from a target sample and an additional sample, respectively, and second sequence information of a nucleic acid fragment from a mixed sample in which the target sample and the additional sample are mixed; Calculating allelic frequency from each of the obtained first sequence information and second sequence information; And comparing the calculated allele frequency for a particular locus of the chromosome to analyze the degree of cross contamination of the sample with respect to the target sample.

According to another aspect, there is provided a nucleic acid probe comprising: a sequence information obtaining unit for obtaining first sequence information of a nucleic acid fragment from each of a target sample and an additional sample, and second sequence information of a nucleic acid fragment from a mixed sample containing the target sample and the additional sample; An allele frequency calculating unit for calculating an allele frequency from the obtained first sequence information and second sequence information, respectively; And an arithmetic unit for comparing the calculated allele frequency with respect to a specific site of the chromosome. The apparatus for analyzing the degree of cross contamination of a sample with respect to a target sample is provided.

Another aspect provides a computer readable recording medium on which a program for executing the above method is recorded.

According to an aspect, there is provided a method for detecting a nucleic acid fragment, comprising: obtaining first sequence information of a nucleic acid fragment from a target sample and an additional sample, respectively, and second sequence information of a nucleic acid fragment from a mixed sample in which the target sample and the additional sample are mixed; Calculating allelic frequency from each of the obtained first sequence information and second sequence information; And comparing the calculated allele frequency for a particular locus of the chromosome to analyze the degree of cross contamination of the sample with respect to the target sample.

The sample may be a biological sample or compound of the subject, i.e., a synthetic sample. The subject may include primates and humans, for example, humans, non-human primates, cows, horses, pigs, sheep, goats, dogs, cats or rodents. The biological sample may be obtained from blood, plasma, serum, urine, saliva, mucous secretion, sputum, feces, tears or a combination thereof. The biological sample of the subject may be a sample of eukaryotic cells, prokaryotes, viruses, bacteriophages, etc. derived from various species. In addition, the sample may include a nucleic acid of the subject or a synthetic nucleic acid. The nucleic acid can be used interchangeably with polynucleotides or oligonucleotides having any length. The nucleic acid may be a cell-free DNA (cf DNA), or may be a separate DNA.

The method of separating the nucleic acid from the sample can be carried out by a method known to a person skilled in the art. The length of the isolated nucleic acid fragment may be from about 10 bp (base pair) to about 2000 bp, from about 15 bp to about 1500 bp, from about 20 bp to about 1000 bp, from about 20 bp to about 500 bp, or from about 20 to about 200 bp.

The step of obtaining the sequence information of the nucleic acid fragment may include the step of performing next-generation sequencing (NGS) on the separated nucleic acid to obtain the sequence information. The "next generation sequencing" can be used interchangeably with "massive parallel sequencing" or second-generation sequencing. Generation sequencing refers to a technique of fragmenting a full-length genome in a chip-based and PCR-based paired end format and performing sequencing at a very high rate based on hybridization of the fragment. Next-generation sequencing is a technique for simultaneously sequencing large quantities of nucleic acids of a fragment, and can perform next-generation sequencing-based targeted sequencing or panel sequencing. Generation sequencing can be performed, for example, on a 454 platform (Roche), GS FLX Titanium, Illumina MiSeq, Illumina HiSeq, Illumina Genome Analyzer, Solexa platform, SOLiD System (Applied Biosystems), Ion Proton (Life Technologies), Complete Genomics, Helicos Biosciences Heliscope , The Single Molecular Real Time (SMRT (TM)) technology of Pacific Biosciences, or a combination thereof.

The method may further comprise preparing a nucleic acid library to perform next generation sequencing. The nucleic acid library can be prepared according to the next generation sequencing method. A nucleic acid library can be prepared according to the manufacturer's instructions to provide next generation sequencing.

The sequence information of the obtained nucleic acid fragment can be referred to as a read.

The sequence information of the nucleic acid fragment can be stored in the system and N-masking can be performed. The N masking means treating the individual nucleic acid read with an excessively low quality as a missing value. In addition, a low-quality read filter can be performed. The low-quality lead filter means that the sequence information of the nucleic acid fragment read at an excessively low quality is processed to be excluded from the analysis.

The method may include mapping the obtained sequence information to a human reference genome to designate the sequence information of the nucleic acid fragment to a chromosome. The human reference genome may be hg18 or hg19. Sequence information mapped to only one genome position in the human reference genome can be designated as unique sequence information. Sequence information of the nucleic acid fragment can be assigned to the position of the chromosome based on the designated unique sequence number. The site of the chromosome may be a continuous range on a chromosome having a length of about 5 kb, about 10 kb, about 20 kb, about 50 kb, about 100 kb, about 1000 kb, or 2000 kb or more. The chromosomal locus may be a single chromosome.

In mapping the obtained sequence information to a human reference genome, a global alignment or a local alignment may be performed in parallel. The global alignment means a method of locating the whole sequence information of the nucleic acid fragment at the most similar part of the reference genome and the local sorting method is a method of locating a part of the sequence information of the nucleic acid fragment at the most similar part of the reference genome sequence do.

The method may comprise identifying a variation in the DNA of the sample. The mutation confirmation can be performed using known mutation detection programs such as GATK, SAMtool, MoDIL, SeqSeq, PeMer, Variation Hunter, Pindel, BreakDancer and Mutek, but is not limited thereto.

The first sequence information may be sequence information of the nucleic acid fragments obtained from each of the plurality of samples including the target sample and the additional sample. The first sequence information may be a result of sequencing the target sample alone. In addition, the first sequence information may be a result of performing sequencing of each sample separately for one or more, two or more, or five or more additional samples.

The second sequence information may be sequence information of the nucleic acid fragment obtained from the mixed sample in which the target sample and the additional sample are mixed. The sequencer performing the sequencing can be a mixed sample in which a plurality of samples are mixed. In the case of using a mixed sample in which a plurality of samples are mixed, there is an advantage in that the cost in the enrichment step of the target is reduced and the high throughput is provided in a short time. At this time, a plurality of samples can be distinguished from each other by tagging a unique mark to each of a plurality of sample libraries.

The method may comprise calculating allelic frequency from each of the obtained first sequence information and second sequence information, respectively. In the target region subjected to sequencing, the allele frequency of each allele can be calculated. The allele frequency may refer to a numerical value indicating the proportion of different alleles constituting the same gene in a sample. The allele frequency can be represented by the frequency of one or more of A, G, C, and T, or the sequence information of all of A, G, C, and T.

The method may comprise comparing the calculated allele frequency for a particular locus of the chromosome.

The specific position of the chromosome may be the same exon or intron spot among the plurality of samples and may be the same number of digits on the same number of chromosomes.

For each specific site of the same target sample and chromosome, the respective allele frequency can be compared from the first sequence information to the respective allele frequency and the second sequence information. For example, the allele frequency of A from the first sequence information and the allele frequency of A from the second sequence information can be compared for a specific site of the same target sample and chromosome. Likewise, the frequencies of alleles of G, C, and T, respectively, from the allele frequency of each of G, C, and T from the first sequence information and the second sequence information can be compared with respect to specific positions of the same target sample and chromosome. As a result of comparison, if there is a significant difference in the allele frequency of any of A, G, C, or T, the target sample may be judged to be contaminated by the additional sample. The larger the significant difference, the more likely that the particular spot in the target sample is more contaminated by the additional sample. The number of alleles having the above-mentioned allele frequency may be compared with the number of alleles having the frequency of alleles or the total number of alleles may be compared with the number of alleles having the corresponding allele frequency.

The "cross-contamination of the sample" means that the tagged tagged in the sequence information of the nucleic acid fragment of another sample is tagged in the sequence information of the nucleic acid fragment of one sample, or the sequence information of the nucleic acid fragment of different samples By exchanging tags in the liver, it means that sequence information of the nucleic acid fragments to which the tag is wrongly tagged is generated. Due to the cross contamination of the sample, when the allele frequency was analyzed from the first sequence information and the allele frequency was analyzed from the second sequence information, the allele frequency was significant It can show a difference.

The method comprises the steps of: selecting, in the obtained first sequence information, a set of mutation prediction positions by combining mutation prediction positions obtained from the sequence information of each of the target sample and the additional sample; ; Calculating an allele frequency of a genotype allele and a background allele from the first sequence information and the second sequence information on the mutation predicted spot set or the control group spot set; And comparing the calculated allele frequencies with respect to the mutation prediction set or control set.

The mutation may refer to different characteristics of a plurality of samples appearing at a specific site of a chromosome. The characteristic may be a nucleic acid sequence. For a particular locus of a chromosome, the genotype allele of any one of the samples obtained from the first sequence information may have a different nucleic acid sequence from the genotype allele of the other sample obtained from the first sequence information. The mutation may be a single nucleotide polymorphism (SNP). SNP refers to a genetic change or mutation that shows differences in the nucleotide sequence (A, G, C, T) of a specific site in a DNA nucleic acid sequence, and is a variation of a single nucleic acid difference between single species of individuals . In particular, SNP is a genetic component associated with disease, and the difference in SNP results in different disease resistance, sensitivity, and degree of disease for each subject. Each of the plurality of samples may have different or identical SNP positions.

The variation may be a variation with respect to the reference dielectric. Specifically, the mutation may comprise a variation of the nucleic acid sequence relative to the reference genome. Variations of the nucleic acid sequence may include substitution, insertion, deletion, or translocation of one or more nucleotide sequences relative to the reference genome. The substitution of the one or more nucleotide sequences may be, for example, Single Nucleotide Variation (SNV). SNV refers to the difference between single bases occurring in a single sequence or a small group of species within a species, for example, the difference from the sequence of the reference genome that appears in the sequencing data. Each of the plurality of samples may have different or SNV positions. The allele frequency of a mutation can be calculated by counting the number of each allele using an existing program such as samtools in the next generation sequencing data.

The method comprises the steps of: selecting a set of mutation prediction sites by combining mutation prediction sites obtained from the sequence information of each of the target sample and the additional sample; and selecting a place excluding the mutation prediction site set as a control site set.

The "variation prediction site" may refer to a specific site of a chromosome having the mutation described above. May refer to the place where the genotype allele of any one of the samples obtained from the first sequence information differs from the genotype allele of another sample obtained from the first sequence information. The place may be a predictor of variation of the sample. For example, if the genotype allele of the target sample has a SNP, the SNP site may be included in the predicted position of the target sample. Each of the plurality of samples may have mutually different or similar mutation prediction sites. Referring to FIG. 3, for the 1 st, 2 nd, 3 rd, 4 th and 5 th digits, the predicted place for the variation is 2 to 4 for the sample 1 (S 1) The seat can be numbered 2 to 5.

The "union variant set" is a set of mutation prediction positions obtained by combining the plurality of samples, that is, the mutation prediction positions of each of the target sample and the additional sample, as the union variance set of the plurality of samples. Lt; / RTI > Referring to FIG. 3, for the 1 st to 5 th digits, the predicted place set of the samples 1 and 2 may be 2 to 5 digits.

A place excluding the set of the predicted shifts may be selected as the set of the control group. Since the background alleles of the plurality of samples obtained in the first sequence information are the same for a specific site of the chromosome, the "control site set" is a set of segregations in which no mutation is detected in any of the plurality of samples .

The method can calculate the allele frequency of the alleles, that is, the genotype allele and / or the allele of the background allele from the first sequence information and the second sequence information obtained for the mutation prediction set group or the control group set, have. Of allelic frequencies calculated from the first sequence information and the second sequence information described above, the allele frequency of the genotype allele and / or the background allele to the mutation predictor set or the control group set can be selected. The allele frequency of the target sample can be represented by the frequency of one or more of A, G, C, and T or the sequence information of all of A, G, C, and T.

Wherein the allele obtained from the first sequence information has an allele frequency of less than 10%, the allele is determined as a background allele, and if the allele has an allele frequency of 10% or more, The gene can be determined as a genotype allele. The criteria for distinguishing the allele may be any criterion for genotyping the genotype.

The "background allele" may refer to an allele having an allele frequency of less than 10%, 5%, 1%, 0.5% or 0.1% in the sequence information.

The "genotype allele" may refer to an allele having an allele frequency of 10% or more obtained from the sequence information. The allele frequency of the genotype allele may be greater than 10%, greater than 30%, greater than 50%, greater than 90%, or 100%. For a specific chromosomal locus, a total of four alleles, typically A, G, C and T, may be present, and a nucleic acid sequence having an allele frequency of 10% or more may be used as a genotype allele, May be determined as a background allele. Referring to FIG. 3, the genotype allele at position 1 of sample 1 is denoted by T, wherein the background alleles are A, G, and C, respectively. In addition, the genotype allele at position 5 of sample 1 is denoted by T and C, and the background alleles are A and G, respectively.

The method may comprise comparing the calculated allele frequencies for a set of mutation predicted sites or a set of control sites.

The allele frequency can be compared in the first sequence information and the second sequence information with respect to the same target sample and the set of predicted displacement. For example, with respect to the same target sample and a set of predicted positions of mutation, the allele frequency of A in the second sequence information and the allele frequency of A in the first sequence information can be compared. Similarly, the allele frequency of each of G, C, and T in the first sequence information and the allele frequency of each of G, C, and T in the second sequence information can be compared with respect to the same target sample and the predicted sequence of the mutation. As a result of the comparison, if there is a significant difference in the allele frequency of any of A, G, C, or T, the target sample may be judged to be contaminated by another sample.

For the same target sample and the control site set, the allele frequency can be compared in the first sequence information and the second sequence information. For example, for the same target sample and control set, the allele frequency of A in the second sequence information and the allele frequency of A in the first sequence information can be compared. Similarly, for all of the same target sample and control set, the allele frequency of each of G, C and T in the second sequence information and the allele frequency of each of G, C and T in the first sequence information can be compared. At this time, since the background allele and the genotype allele of the plurality of samples obtained from the first sequence information are the same for the control site set, it can be judged that there is no cross contamination of the sample. Referring to FIG. 3, the first position of all the samples is a place where the genotype allele is the same as T, the background allele is the same as A, G and C, and the mutation is not detected. The first digit is a place in the control set. It can be judged that the background allele of any one sample is not interfered with by the genotype allele of another sample.

The method comprises selecting for a set of mutation predicting place alleles which are both a background allele of the target sample and a genotype allele of the additional sample in the first sequence information into a test group, , Selecting alleles which are background alleles of the target sample and background alleles of the additional sample in the first sequence information as a control group.

The "control group" means an allele, which is the background allele of the target sample and the background allele of the additional sample in the first sequence information with respect to the mutation predicted spot set and the control group spot set.

The method may include comparing the allele frequency of the control group obtained from the target sample in the first sequence information and the allele frequency of the control group obtained from the target sample in the second sequence information.

3, the background allele of the first spot of the sample 1 (S 1) and the background allele of the sample 2 (S 2), the sample 3 (S 3), and the sample 4 (S 4) Alleles that are genes are A, G, and C. In the obtained first sequence information, the allele frequency of A, G and C as the control group of sample 1 and the allele frequency of A, G and C of the control group of sample 1 can be compared with each other in the second sequence information. Alleles which are background alleles at position 2 of sample 1 and at the same time background alleles of sample 2, sample 3 and sample 4 which are additional samples are G and C, respectively. In the obtained first sequence information, the allele frequency of G and C as the control group of sample 1 and the allele frequency of G and C as the control group of sample 1 in the second sequence information can be respectively compared. Also, the allele of the background allele of sample 1, the background allele of sample 1, and the background allele of sample 2, sample 3, and sample 4, which are additional samples, is A at the same time. In the obtained first sequence information, the allele frequency of the control group A of the sample 1 and the allele frequency of the control group A of the sample 1 can be compared with each other in the second sequence information. The control group of these target samples may have little or no difference when the allele frequency in the first sequence information is compared with the allele frequency in the second sequence information. The control group can judge that there is no possibility of cross contamination of the sample.

The "test group" means an allele, which is the background allele of the target sample and the genotype allele of the additional sample, in the first sequence information with respect to the set of mutation prediction positions. Since the test group is judged to have a possibility of cross contamination of a sample at a chromosome specific site corresponding to a plurality of samples, the degree of contamination can be analyzed.

The method may compare the allele frequency of the test group obtained from the target sample obtained from the target sample among the first sequence information and the allele frequency of the test group obtained from the target sample among the second sequence information.

Depending on how the sample is mixed, the method of analyzing the degree of contamination and selecting the test group may vary. Samples and chromosomes may be different if contamination occurs at specific sites. If cross-contamination occurs between samples of a target sample, the allele frequency of the background allele in the set of predicted shifts of the target sample may be affected by genotype alleles of other samples. The comparing step may analyze the number of alleles having an arbitrary allele frequency in the test group and / or the control group. The number of alleles having the allele frequency may be compared with the number of alleles having the frequency of alleles or the number of alleles having the frequency of alleles may be compared with the total number of alleles.

Referring to FIG. 3, alleles which are background alleles at position 4 of sample 1 and at the same time additional samples, sample 2, sample 3, and sample 4, which are genotype alleles, are T. In the obtained first sequence information, the allele frequency of T, which is the test group of sample 1, and the allele frequency of T, which is the test group of sample 1, can be compared with the second sequence information. In addition, the allele gene which is the background allele of the 4th position of the sample 2 and the additional alleles of the sample 1, the sample 3 and the sample 4, which is a genotype allele, is G. In the obtained first sequence information, the allele frequency of the test group G of the sample 2 can be compared with the allele frequency of the test group G of the sample 2 in the second sequence information. Since the allele frequency of background allele G at position 4 in sample 2 can be influenced by genotype allele G of sample 1, sample 3 and sample 4, the allele frequency of background allele G in sample 2 is It can be different. In addition, if it is the background allele of the second position of the sample 1, the allele of the additional alleles of the sample 2, the sample 3 and the sample 4 is T. In the obtained first sequence information, the allele frequency of T, which is the test group of sample 1, and the allele frequency of T, which is the test group of sample 1, can be compared with the second sequence information.

In another aspect, there is provided a nucleic acid amplification apparatus comprising: a sequence information obtaining unit for obtaining first sequence information of a nucleic acid fragment from a target sample and an additional sample, respectively, and second sequence information of a nucleic acid fragment from a mixed sample in which the target sample and the additional sample are mixed; An allele frequency calculating unit for calculating an allele frequency from the obtained first sequence information and second sequence information, respectively; And an arithmetic part for comparing the calculated allele frequency with respect to a specific site of the chromosome. The apparatus 100 is provided for analyzing the cross contamination degree of a sample with respect to a target sample.

The apparatus may comprise a " part "or" ... "module that implements the method of analyzing the degree of cross contamination of the sample in a time-series manner. Therefore, even if omitted below, the method described above for analyzing the cross-contamination degree of the sample can be applied to the apparatus for analyzing the degree of cross-contamination of the sample. The components may correspond to a processor. Thus, such a processor may be implemented as an array of logic gates, and may be implemented as a combination of a general purpose microprocessor and a memory in which a program executable in the microprocessor is stored. It will be appreciated by those of ordinary skill in the art that other types of hardware may also be implemented.

The sequence information obtaining unit 110 obtains sequence information from the sequencing device. The calculating unit 120 analyzes the allele frequency from the obtained first sequence information and second sequence information, respectively. The calculation unit compares the allele frequency calculated from the first sequence information and the second sequence information with respect to a specific spot of the chromosome. The operation unit 130 may compare the number of alleles having the allele frequency by the allele frequency, or may compare the ratio of the number of alleles having the allele frequency with the total number of alleles.

Wherein the apparatus comprises: a means for selecting, as a set of mutation prediction positions, a set of mutation prediction positions obtained by combining the mutation prediction positions obtained from the sequence information of each of the target sample and the additional sample in the obtained first sequence information, A seat for selecting a seat; An allelic frequency calculating unit for calculating the allele frequency of the genotype allele and the background allele from the first sequence information and the second sequence information for the mutation predicted spot set or the control group spot set; And an arithmetic unit for comparing the calculated allele frequency with respect to the set of the predicted shift positions or the set of the control group.

The digit selection unit 140 selects a set of predicted shift positions by combining the predicted positions of each of the plurality of samples and selects a control group set by combining the positions where no variation is detected in any of the plurality of samples .

The apparatus may include a group selection unit for selecting a test group and a control group based on the set of the predictive shift positions and the set of control sequences. The group selector 150 selects a test group and a control group.

And an allelic frequency calculating unit for calculating the allele frequency of the genotype allele and the background allele from the first sequence information and the second sequence information obtained for the mutation predicted spot set or the control group spot set. The allele frequency calculating unit may calculate the allele frequency of an allele including a genotype allele and / or a background allele.

The group selector may select alleles that are both a background allele of the target sample and a genotype allele of the additional sample as test groups in the first sequence information, For the set, alleles which are both the background allele of the target sample and the background allele of the additional sample in the first sequence information can be selected as a control group. If necessary, the test group and the control group can be selected at the same time or sequentially.

The arithmetic unit compares the allele frequency of the test group obtained from the target sample among the first sequence information and the allele frequency of the test group obtained from the target sample among the second sequence information and obtains from the target sample among the first sequence information The allele frequency of the control group obtained from the target sample and the allele frequency of the control group obtained from the target sample among the second sequence information. The arithmetic unit may analyze the number of alleles having an arbitrary allele frequency in the test group and / or the control group. The number of alleles having the allele frequency may be compared with the number of alleles having the frequency of alleles or the number of alleles having the frequency of alleles may be compared with the total number of alleles.

Wherein the alleles obtained from the first sequence information have an allele frequency of less than 10%, the alleles are determined as background alleles, and if the allele has an allele frequency of 10% or more, And an allele determining unit 160 for determining the gene as a genotype allele.

Another aspect provides a computer-readable recording medium on which a program for executing a method for analyzing the degree of cross contamination of a sample with respect to the target sample is recorded.

The method may be implemented in the form of software readable by various computer means and recorded in a computer-readable recording medium. Here, the recording medium may include program commands, data files, data structures, and the like, alone or in combination. The program instructions to be recorded on the recording medium may be those specially designed and constructed for the method according to the above, or may be available to those skilled in the art of computer software.

For example, the recording medium may be an optical recording medium such as a magnetic medium such as a hard disk, a floppy disk and a magnetic tape, a compact disk read only memory (CD-ROM), a digital video disk (DVD) Includes a hardware device that is specially configured to store and execute program instructions such as a magneto-optical medium such as a floppy disk and a ROM, a random access memory (RAM), a flash memory, do. Examples of program instructions may include machine language code such as those generated by a compiler, as well as high-level language code that may be executed by a computer using an interpreter or the like. Such a hardware device may be configured to operate as one or more software modules to perform the operations of the above-described method, and vice versa.

Although the present specification and drawings describe exemplary device configurations, the functional operations and subject matter implementations described herein may be embodied in other types of digital electronic circuitry, or alternatively, of the structures disclosed herein and their structural equivalents May be embodied in computer software, firmware, or hardware, including, or in combination with, one or more of the foregoing. Implementations of the subject matter described herein may be embodied in one or more computer program products, that is, a computer program product encoded on a type of program storage medium for execution by, or in control of, And can be implemented as a module as described above. The computer-readable medium can be a machine-readable storage device, a machine-readable storage substrate, a memory device, a composition of matter that affects the machine readable propagation type signal, or a combination of one or more of the foregoing.

A computer program (also known as a program, software, software application, script, or code) that is embedded in the apparatus according to the above method and that executes the method may be any of a compiled or interpreted language, a programming language including a priori or procedural language And may be deployed in any form including stand-alone programs or modules, components, subroutines, or other units suitable for use in a computer environment. A computer program does not necessarily correspond to a file in the file system. The program may be stored in a single file provided to the requested program, or in multiple interactive files (e.g., a file storing one or more modules, subprograms, or portions of code) (E.g., one or more scripts stored in a markup language document). A computer program may be deployed to run on multiple computers or on one computer, located on a single site or distributed across multiple sites and interconnected by a communications network.

Sequence information is obtained from a plurality of biological samples in which individual samples are mixed, and in the process of extracting mutations, the contamination ratio at the chromosomal site can be accurately measured when the sample is contaminated. The influence of cross contamination between conventional samples is ignored or compared with numerical values of known databases. However, since the degree of contamination between samples can be measured using numerical values of experimental results obtained in the platform of the experiment , Reliability can be given to the mutation extraction result of the individual samples. Further, in analyzing similar samples, the degree of cross-contamination of the sample that can be generated by the protocol used for analysis can be normalized.

FIG. 1 is a diagram for explaining a method of selecting a set of prediction prediction positions.
Figure 2 is a graph showing the proportion of the number of background alleles having an allele frequency of 0 to 0.01 in the test group and the control group.
3 is a diagram for explaining a method of selecting a control group and a test group among a plurality of samples.
4 is a block diagram showing a configuration of an apparatus for analyzing the degree of cross contamination of a sample.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

One. Hapmap  Mutation extraction from cell line

Eight normal HapMap cell lines were purchased from the Coriell Institute (http://ccr.coriell.org/). DNA concentration and purity of the cell line were measured by Picogreen fluorescence analysis using a Nanodrop 8000 UV-Vis spectrometer (Thermo Scientific) and Qubit 2.0 fluorescence photometer (Life Technologies) Respectively. Slice size distributions representing the extent of DNA degradation were measured using a 2200 TapeStation instrument (Agilent Technologies) and real-time PCR Mx3005p (Agilent Technologies) using the manufacturer's instructions.

The gDNA of the cell line was subjected to sonication using Covaris S2 (7 min, 0.5% duty, intensity = 0.1, 50 cycles / burst; Covaris Inc.) and sectioned into about 150 to about 200 bp Respectively. The fragments were then purified using a 1.8-fold volume of AMPure XP beads (Beckman Coulter) of the gDNA samples. After fragmentation, end-pair, A-tailing, adopter ligation, and PCR reactions were performed in KAPA Hyper kit (Kapa), before enrichment of the target Biosystem Inc.). Ligation was performed overnight at 4 ° C using Pentabase indexed adapters as an adapter.

Agilent SureDesign was used to design a unique RNA bait targeting ~ 0.5 Mb of the human genome. The genome contains an exon from 83 cancer-related genes that are frequently rearranged in solid tumors and an intron from 5 genes. After library of cell line samples was pre-amplified, double stranded DNA concentrations were measured using a Qubit Fluorometer (Life Technologies). The section size distribution was measured using a 2200 TapeStation instrument (Agilent Technologies). The library was adjusted to a total of 750 ng of DNA for each hybridization selection reaction. SureSelect's blocking oligonucleotides were used for hybridization selection.

Prior to capture hybridization, based on DNA concentration and average slice size, the library was labeled to be distinguishable for each of a plurality of samples, and each library was normalized to the same concentration of 2 nM and pooled to the same volume. After denaturation of the library with 0.2N NaOH, the library was diluted to 20 pM. Cluster amplification of the denatured template was performed and the flowcells were sequenced using a HiSeq 2500 v3 Sequencing-by-Synthesis Kit (2x100 bp lead) followed by RTA v. 0.12. Base was extracted using 4.2.

The resulting leads were aligned to the hg19 human reference using BWA v0.7.5a35 to obtain BAM files. SAMtools v0.1.18 36, GATK v2.2-2537, and Picard v1.93 can be used to classify local realignment, duplicate markings, and SAM / BAM to identify out-of-target leads, , And redundancy was removed. Mutations were then detected using MuTect 1.1.4.

2. Of the sample  Check the degree of cross contamination

Sequence information was obtained and the test group and control group were selected from the calculated allele frequencies as follows. Allelic frequencies of background alleles were confirmed in each group. Within the allele frequency interval of 0 to 0.01, the number of background alleles having an allele frequency in this interval was confirmed. The ratio of the number of background alleles with the allele frequency in the total number of background alleles by group was calculated. At this time, the allele frequency of 1% or less was determined as the background allele, and the allele frequency of 10% or more was determined as the genotype allele.

Among each group
Have the allele frequency
Average ratio of alleles Among each group
Have the allele frequency
Number of alleles Allele frequency
Interval Exclusive
Test
group 8 species mixed
Test
group Exclusive
Control group
group 8 species mixed
Control group
group Exclusive
Test
group 8 species
Test
group Exclusive
Control group
group 8 species
Control group
group 0 0.797848869 0.669169306 0.919425121 0.925068092 373155 312971 1291528 1299454 0.001 0.088772281 0.151291505 0.055996532 0.053292686 41519 70759 78659 74861 0.002 0.049208593 0.076875978 0.016010577 0.014389426 23015 35955 22490 20213 0.003 0.024514934 0.045659392 0.005270831 0.004536428 11466 21355 7404 6372 0.004 0.019319821 0.025120766 0.001901991 0.001561708 9036 11749 2672 2194 0.005 0.010829032 0.012480406 0.000716695 0.000587665 5065 5837 1007 826 0.006 0.002457644 0.006990689 0.000303265 0.000244267 1149 3270 426 343 0.007 0.001761265 0.004264514 0.000143802 0.000120398 824 1995 202 169 0.008 0.001060489 0.003364711 7.51044E-05 6.83414E-05 496 1574 106 96 0.009 0.000359712 0.001290674 4.62728E-05 3.94209E-05 168 604 65 55 0.01 0.000700776 0.000709409 2.86536E-05 2.45602E-05 328 332 40 35 0.011 0.001408678 0.00036075 1.44158E-05 1.41488E-05 659 169 20 20 0.012 0 0.000180375 1.47717E-05 1.03224E-05 0 84 21 15 0.013 0 0 1.06783E-05 6.40701E-06 0 0 15 9 0.014 0 0.000180375 5.69512E-06 5.7841E-06 0 84 8 8

- Allele frequency values less than 0.014 are omitted.

2-1. Identification of allele frequency distribution in test group

In the result of obtaining the sequence information of any one of the haplotype cell samples, it was confirmed that 467,701 alleles were included in the test group. The number of alleles having an allele frequency in the allele frequency interval of 0 to 0.01 was analyzed for the test group in the result of performing sequencing on the mock matrix cell sample alone and the ratio thereof was shown in the graph 1 and 2, single test group). In addition, the number of alleles having an allele frequency in the allele frequency interval of 0 to 0.01 was calculated for the test group by sequencing with a mixed sample of eight kinds of mapped cell lines containing the corresponding mapped cell line sample (See Table 1 and Fig. 2, 8-plex, Test group).

Referring to FIG. 2 and Table 1, it can be seen that the number of alleles having a specific background allele frequency (allele frequency of background allele) is different. For example, the group with an allele frequency of 0.007 in the test group was found to be about 0.176% when analyzed alone and about 0.427% when analyzed in a mixture of eight samples. When analyzed in single or mixed samples of 8 species, the allele frequency of background allele was changed even in the same mock-up cell line sample.

The average allele frequency was determined by dividing the product of the background allele frequency and the number of alleles having the background allele frequency by the total number of alleles belonging to the test group. Referring to FIG. 2, the average background allele frequency of the mock-up cell line samples was about 0.052% when the mock-up cell line samples were analyzed alone. The average background allele frequency of the mock-up cell line samples was about 0.077% when analyzed in mixed samples containing 8 hap cell line samples. Therefore, it can be seen that the sample cell line has an average contamination level of about 0.025% by the other mock-up cell line samples.

2-2. Identification of the allele frequency distribution of the control group

As a result of obtaining the sequence information of any one of the mosaic cell line samples, it was confirmed that 1,404,712 alleles were included in the control group. The number of alleles having an allele frequency in the allele frequency interval of 0 to 0.01 was analyzed for the test group in the result of performing the sequencing by the sample myeon cell line alone, and the ratio thereof was shown in the graph 1 and 2, single, control group). In addition, the number of alleles having an allele frequency in the allele frequency interval of 0 to 0.01 was calculated for the test group by sequencing with a mixed sample of eight kinds of mapped cell lines containing the corresponding mapped cell line sample , And the ratio is shown in the graph (see Table 1 and Fig. 2, 8-plex, control group). Referring to FIG. 2 and Table 1, it can be seen that the number of alleles having a specific background allele frequency is not substantially different. For example, the group with an allele frequency of 0.007 in the control group was about 0.014% when analyzed alone and about 0.012% when analyzed in a mixed sample of 8. When analyzed in single or mixed samples of 8 species, it was confirmed that there was little difference in allele frequency of background allele in the same mock-up cell line sample.

The average allele frequency was determined by dividing the product of the background allele frequency and the number of alleles having the background allele frequency by the total number of alleles belonging to the control group. Referring to FIG. 2, the average background allele frequency of the mock-up cell line samples was about 0.012% when the mock-up cell line samples were analyzed alone. The mean background allele frequency of the mock-up cell line samples was about 0.011% when analyzed in mixed samples containing 8 hap cell line samples. Therefore, it was confirmed that the control group of the haplotype cell line samples had little or no influence of contamination by other haplotype cell line samples.

The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.

Claims (15)

In a computer-based system, the sequential information obtaining means equipped with a computer is capable of obtaining first sequence information of a nucleic acid fragment from each of a target sample and an additional sample, and second sequence information of a nucleic acid fragment from a mixed sample containing the target sample and the additional sample ;
Calculating allelic frequency from each of the first sequence information and the second sequence information obtained by the calculating means; And
CLAIMS 1. A method for analyzing the degree of cross contamination of a sample with respect to a target sample, comprising comparing the calculated allele frequency with a specific site of the chromosome,
Wherein the comparing step comprises the steps of: selecting, in the obtained first sequence information, a set of mutation prediction positions by combining mutation prediction positions obtained from the sequence information of each of the target sample and the additional sample; Selecting a set of control seats;
Obtaining an allelic frequency of a genotype allele and a background allele from the first sequence information and the second sequence information for the mutation predicted spot set or the control group spot, respectively; And
Comparing the obtained allele frequency for the mutation predicted spot set or the control set set,
Wherein the background allele is determined as a background allele when the allele obtained from the first sequence information has an allele frequency lower than a predetermined reference value,
Wherein the allele of the genotype allele is determined as a genotype allele when the allele obtained from the first sequence information has an allele frequency equal to or higher than a predetermined reference value. delete The method according to claim 1,
Wherein the selecting comprises selecting alleles that are both the background allele of the target sample and the genotype allele of the additional sample in the first sequence information as a test group,
Selecting alleles that are background alleles of the target sample and background alleles of the additional sample in the first sequence information as a control group, for the mutation predicting place set and the control group place set. The method of claim 3,
The step of comparing the allelic frequencies obtained comprises comparing the allele frequency of the test group obtained from the target sample obtained from the target sample among the first sequence information and the allele frequency of the test group obtained from the target sample among the second sequence information ≪ / RTI > The method of claim 3,
Comparing the obtained allele frequency comprises comparing the allele frequency of the control group obtained from the target sample among the first sequence information with the allele frequency of the control group obtained from the target sample of the first sequence information and the allele frequency of the control group obtained from the target sample among the second sequence information ≪ / RTI > The method according to claim 1,
Wherein the background allele is determined as a background allele when the allele obtained from the first sequence information has an allele frequency of less than 10%
Wherein the genotype allele is a genotype allele when the allele obtained from the first sequence information has an allele frequency of 10% or more. 2. The method of claim 1, wherein the variation is a SNP or SNV. A sequence information obtaining unit for obtaining first sequence information of a nucleic acid fragment from each of the target sample and the additional sample and second sequence information of the nucleic acid fragment from a mixed sample containing the target sample and the additional sample;
An allele frequency calculating unit for calculating an allele frequency from the obtained first sequence information and second sequence information, respectively; And
And a calculation unit for comparing the calculated allele frequency with respect to a specific site of the chromosome,
An apparatus for analyzing the degree of cross contamination of a sample with respect to a target sample,
Wherein the arithmetic unit selects, in the obtained first sequence information, a set of mutation prediction positions by combining mutation prediction positions obtained from the sequence information of each of the target sample and the additional sample, A seat for selecting a seat;
An allelic frequency obtaining unit for obtaining the allele frequency of the genotype allele and the background allele from the first sequence information and the second sequence information obtained for the mutation predicted spot group or the control group spot; And
And an arithmetic unit for comparing the obtained allelic frequency with respect to the mutation prediction set or control set,
Wherein the background allele is determined as a background allele when the allele obtained from the first sequence information has an allele frequency lower than a predetermined reference value,
Wherein the genotype allele comprises an allele determining unit that determines the allele as a genotype allele when the allele obtained from the first sequence information has an allele frequency equal to or higher than a predetermined reference value. delete The method of claim 8,
Wherein the position selecting unit selects alleles which are both the background allele of the target sample and the genotype allele of the additional sample in the first sequence information as a test group,
And a group selector for selecting, as a control group, alleles which are background alleles of the target sample and background alleles of the additional sample in the first sequence information, with respect to the mutation predicting place set and the control group place set. The method of claim 10,
The arithmetic unit for comparing the obtained allele frequencies includes an allele frequency of the test group obtained from the target sample in the first sequence information and an allele frequency of the test group obtained from the target sample in the second sequence information, / RTI > The method of claim 10,
The arithmetic unit for comparing the obtained allelic frequencies includes an allele frequency of the control group obtained from the target sample in the first sequence information and an allele frequency of the control group obtained from the target sample in the second sequence information, / RTI > The method of claim 8,
Wherein the allele determining unit determines that the allele of the background allele is a background allele when the allele obtained from the first sequence information has an allele frequency of less than 10%
Wherein the genotype allele is a genotype allele when the allele obtained from the first sequence information has an allele frequency of 10% or more. 9. The apparatus of claim 8 wherein the variation is SNP or SNV. A computer-readable recording medium having recorded thereon a program for executing the method according to any one of claims 1 to 3.

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