KR101533792B1 - Method for Autosomal Analysing Human Subject of Analytes based on a Next Generation Sequencing Technology - Google Patents

Abstract

The present invention relates to a method for analyzing an autosome of a human subject based on a next generation sequencing (NGS) technology, wherein the method comprises the steps of: (a) performing multiplex amplification by using primers that complementarily bind, respectively, to D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, von Willebrand factor A (vWA), D8S1179, Human thyroid peroxidase gene (TPOX), Human fibrinogen alpha chain (FGA), and amelogenin loci of a human subject DNA sample; (b) determining the short tandem repeat (STR) allele of each of the loci by using NGS data of the product of the multiplex amplification in the step (a), and identifying the human subject with a gene. The method using an NGS system according to the present invention can be used for analyzing and interpreting an STR profile from a single-source sample, mixed sample, and decomposed DNA sample in forensic science.

Description

TECHNICAL FIELD [0001] The present invention relates to an autosomal analysis method for human body based on NGS,

The present invention relates to an autosomal analysis method of an NGS-based human object.

Short tandem repeats (STRs), which are mainly used in the forensic field, exist in the non-coding region of the human genome, which is characterized by repeated repeats of 2-7 bp nucleotide sequences . Because STR has a different repeat number of core repeat units for each individual and has a unique value for each individual, STR analyzes are used to identify individual identification and blood relationship. ^{1-3) In the} current forensic science practice, amplification products obtained by polymerase chain reaction (PCR) were separated by capillary electrophoresis (CE) The STR genotype is being analyzed. ³⁾ Multiplex PCR (multiplex PCR) is widely used so that amplification products of several STR genome loci can be obtained at the same time. CE-based assays can be used to identify amplification products with a resolution that can distinguish only one base difference, and amplification products can be detected in automated equipment using primers (primers) with fluorescent markers It can be detected easily and quickly. However, this method is not only able to identify the base sequence of the amplification product, but also has limitations on the number of fluorescent markers available and the size of amplification product. The Sanger-based sequencing method, which is a method for analyzing the existing nucleotide sequence, can accurately obtain the nucleotide sequence information, but it is applied to the research field in which a large amount of DNA sequence information such as personal genome analysis is required It is inefficient in terms of analysis time, labor, and cost. Therefore, there is a demand for a new analysis technique that can obtain DNA sequence information of a large capacity at high efficiency and low cost.

In the mid-2000s, next generation sequencing (NGS) was introduced, which can rapidly generate short-length nucleotide sequences in large amounts for template DNA. ^{4) As} the development of NGS equipment, the improvement of reagents, and the development of bioinformatics techniques, NGS analysis has been used in many research fields because it has various advantages to replace the existing Sanger-based method. ^{5-9) In the} field of forensic science, we apply the new NGS technique to the STR analysis to see what advantages it has compared with the existing CE-based method, especially if the limitations of STR analysis in existing methods can be overcome Attempts have been made, and the results of recent research are being published. ^10-15) However, the method for determining STR alleles from the experimental methods such as sample preparation and library preparation for the nucleotide sequence analysis of the STR amplification product by the NGS technique and the generated NGS data has not yet been firmly established. Therefore, in the lip oil study, the optimal experimental method for generating NGS data from the amplification products obtained by the multi-amplification PCR method, which is mainly used for the STR analysis, and the analysis of the large-capacity NGS data, , And to investigate the usefulness of STR genotyping using NGS for 1: 1 mixed samples as well as single samples.

On the other hand, the sequence information of STR alleles can be generated using the NGS technology, and thereby the sequence variant type, namely single nucleotide polymorphism (SNP) and / or insertion-deletion (INDEL) Information is also available. Some forensic scientists focus on sequence mutations rather than STR alleles because they can distinguish alleles of the same length with sequence variations or other repeating structures. In other words, discriminatory power of STR loci can be increased by utilizing sequence variation in STR [10-17].

Previous studies have investigated the possibility of NGS for forensic STR analysis. In one study, CE-based STR kits were used with four STR markers using the GS Junior platform [10] and 9 STRs using the 454 GS FLX [5] It was used to test only markers. However, there was no in-house multiplex PCR system optimized for NGS analysis of STR. In particular, the amplicon size is more important because the read length needs to be considered, and the lead length that can be read on the NGS platform is the most important factor for accurate STR typing [17, 18]. A single read will contain the repeats of the STR. Thus, the present inventors have focused on generating small size amplicon for the NGS lead length. Many researchers have already pointed out that a size optimized multiplex PCR system for NGS analysis is needed. In this regard, the inventors constructed an in-house developed multiplex PCR system using 18 markers producing small amplicons of 70 bp - 210 bp. The in-house multiplex PCR system can be used for forensic analysis using single samples, mixed samples, and resolved DNA samples.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have now found that forensic gene analysis (NGS) using the next generation sequencing (NGS) technology, and to develop an optimized multiplex PCR system. As a result, the method of accurately and effectively analyzing the autosomal chromosome of a human subject was devised by analyzing the NG allele of the STR marker by analyzing STR allelotype determination, repeating structure of alleles, and nucleotide sequence variation, In particular, multiplex amplification was performed with each non-labeled primer complementarily binding to a specific locus and analysis of large amounts of NGS data obtained from these amplification products resulted in a 1: 1 It was confirmed that analysis of STR genotype was possible in mixed samples. The present inventors also devised a size-optimized multiplex PCR system for NGS analysis using 18 STR markers and capable of generating amplicons of 70 bp to 210 bp in size. The inventors of the present invention have completed the present invention by confirming that they can be applied to artificially decomposed DNA samples as well as single-source samples and mixed samples, and in particular, STR loci can effectively generate STR allele call and STR profiles without loss of information .

Accordingly, an object of the present invention is to provide a method of analyzing an autosomal chromosome of a next generation sequencing-based (NGS) human subject.

It is another object of the present invention to provide a multiplex gene amplification kit for NGS-based next-generation sequencing-based autosomal analysis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

The present inventors have now found that forensic gene analysis (NGS) using the next generation sequencing (NGS) technology, and to develop an optimized multiplex PCR system. As a result, the method of accurately and effectively analyzing the autosomal chromosome of a human subject was devised by analyzing the NG allele of the STR marker by analyzing STR allelotype determination, repeating structure of alleles, and nucleotide sequence variation, In particular, multiplex amplification was performed with each non-labeled primer complementarily binding to a specific locus and analysis of large amounts of NGS data obtained from these amplification products resulted in a 1: 1 It was confirmed that analysis of STR genotype was possible in mixed samples. The present inventors also devised a size-optimized multiplex PCR system for NGS analysis using 18 STR markers and capable of generating amplicons of 70 bp to 210 bp in size. It has been shown that it can be applied to artificially degraded DNA samples as well as single-source and mixed samples, and STR in particular can generate STR allele call and STR profiles effectively without loss of information.

I. NGS-based (next generation sequencing-based) autosomal analysis method 1

According to one aspect of the present invention, there is provided a method of analyzing an autosomal chromosome of an NGS-based next generation sequencing-based human subject, comprising the steps of:

(a) Human DnaS1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, vWA A multiplex amplification using primers complementary to the genetic locus of D8S1179, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), and amelogenin; And

(b) determining an STR (short tandem repeat) allele of the genetic locus using the NGS data of the multiplex amplification product of step (a), and identifying the human object as a gene.

In recent years, in order to overcome the limitations of the capillary electrophoresis (CE) -based STR assay, such as the limited number of simultaneously measurable STR loci using the fluorescence-labeled primer and the maximum size of the STR ampicron, Next generation sequencing (NGS), which is a next-generation sequencing method, has attracted attention as a method.

The present inventors have used 15 STR genetic markers (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX, Left and amelogenin) were analyzed and their efficiency was assessed in STR analysis. A sample amplicon was generated by a multiplex PCR method using a single-source and a 1: 1 mixture of male and female standard DNA, and a DNA library was constructed by splicing the adapter using a multiplex identifier (MID) And DNA sequencing was performed using the Roche GS Junior Platform. The sequencing data of each sample was analyzed by alignment with a pre-constructed reference sequence. Most STR alleles were determined by applying 20% of the two single-source coverage thresholds and 10% of the 1: 1 mixture coverage threshold. The structure of the STR in each allele was precisely determined by analyzing the sequence of the target STR region. The mixing ratio of the mixed samples was determined by analyzing the coverage ratio between the respective alleles of each locus and analyzing the reference / mutation rate of the sequence mutations. As a result, NGS data can be successfully generated using the experimental method of the present invention. In addition, the NGS analysis protocol enables accurate STR allele call and repeat structure determination in each locus. Thus, the method of the present invention using the NGS system will be useful for analyzing and interpreting STR profiles in single-source and mixed samples in forensic studies.

A DNA sample to be analyzed according to the present invention is a DNA sample isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or amniotic fluid including fetal cells, and mixtures thereof , But is not limited thereto.

The DNA sample can be obtained through a conventional method known in the art. For example, DNA is isolated by treating the tissue with a DNA lysis buffer (e.g., tris-HCl, EDTA, EGTA, SDS, deoxycholate, and Triton X and / or NP-40) .

According to one embodiment of the present invention, the DNA sample is a biological sample containing DNA. Meanwhile, the DNA sample may be composed of a single-source sample or a mixture of two or more sources.

Although the method of the present invention can apply DNA isolated from a biological sample, Direct PCR (Direct Polymerase Chain Reaction) involving nucleic acid molecules may be performed using the biological sample directly (see Korean Patent Registration No. 10-0746372).

Hereinafter, the gene detection method of the present invention will be described step by step in detail:

Step (a): Multiplex amplification step

First, the DNA samples to be analyzed include D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, vWA ), D8S1179, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA), and amelogenin.

The term " amplification " as described herein refers to a reaction that amplifies a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202, and 4,800,159), reverse transcription-polymerase chain reaction (RT- (Sambrook et al., Molecular Cloning . A Laboratory Manual , 3rd ed. Methods of ligase chain reaction (LCR) (17, 18), Gap-HI (WO 89/06700) and Davey, C. et al (EP 329,822) (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 19 (WO 88/10315), self sustained sequence replication) 20 (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), consensus sequence primed polymerase chain reaction (CP-PCR) (U.S. Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence-based amplification acid sequence based amplification (NASBA) (U.S. Patent Nos. 5,130,238, 5,4 But are not limited to, ribozymes, ribosomes, ribosomes, ribosomes, ribosomes, ribosomes, ribosomes, ribosomes, and ribosomes. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617 and U.S. Patent No. 09 / 854,317.

PCR is the most well-known nucleic acid amplification method, and many variations and applications thereof have been developed. For example, touchdown PCR, hot start PCR, nested PCR and booster PCR have been developed by modifying traditional PCR procedures to enhance the specificity or sensitivity of PCR. In addition, multiplex PCR, real-time PCR, differential display PCR, D-PCR, rapid amplification of cDNA ends (RACE), inverse PCR chain reaction (IPCR), vectorette PCR, and thermal asymmetric interlaced PCR (TAIL-PCR) have been developed for specific applications. For more information on PCR, see McPherson, MJ, and Moller, SG PCR . BIOS Scientific Publishers, Springer-Verlag New York Berlin, Heidelberg, NY (2000), the teachings of which are incorporated herein by reference.

In the present invention, the multiplex amplification is a multiplex PCR (Polymerase Chain Reaction) amplification or a direct multiplex PCR amplification. According to one embodiment of the present invention, the multiplex amplification is an emulsion multiplex PCR amplification.

According to one embodiment of the present invention, the multiplex PCR amplification has an annealing temperature condition of 55-65- < 0 > C. According to another embodiment of the present invention, the multiplex PCR amplification has a temperature of 57-62 Annealing temperature conditions, and according to certain embodiments of the present invention, the multiplex PCR amplification has an annealing temperature condition of 60 < 0 > C.

The multiplex PCR amplification requires a reasonable number of cycles to perform PCR. According to one embodiment of the invention, the multiplex PCR amplification is performed in 32-36 cycles, 33-35 cycles or 34 cycles.

The present invention relates to a DNA sample which is complementarily binding to the D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX, FGA and Amelozynine genetic loci Multiplex amplification is performed using a primer.

The primer used in the present invention may be used with a fluorescent dye or a separate adapter sequence attached thereto, and may be used as a non-labeled primer.

According to one embodiment of the present invention, the primer used in the present invention is a non-labeled primer, which is a primer that is not labeled with a specific fluorescent dye or has no adapter. Unlike the STR assay using CE, the present inventors did not attach fluorescent markers to the primers. This is because the accuracy of the allele determination and the quality of the NGS data set, as compared with the case where the non-labeled primer is attached with a fluorescent marker or an adapter, ) Were found to be high.

The present inventors prepared three types of primer sets according to the presence or absence of the fluorescent label and the adapter sequence in the primer sequence of Table 1 to generate respective PCR amplification products and performed NGS (fluorescent label: Test 1, 6; Adapter sequence: Test 2, 3; non-labeled: Test 4, 5). As a result, the quantitative aspects (total number of readings) of the data obtained by NGS were about 80,000 in the case of designing the primer including the adapter sequence (Test 2 and 3), while the data set obtained from the non-primer Test 1, 4, 5, 6), about 14 to 150 thousand samples were obtained. As for the distribution according to the read length, Test 1 and 6 show a narrow range around 200 bp, whereas Test 2-5 shows a relatively wide range at 50 bp - 400 bp.

Next, the NGS data set was analyzed using the lobSTR program or the Bowtie 2 program. As a result, in terms of accuracy of allele designation, i) Tests 4 and 5 in which PCR was performed with a primer without fluorescent label and adapter sequence, ii) Tests 1 and 6 in which PCR was performed with a fluorescent label attached primer, iii) The accuracy was high in the order of Test 2 and 3 in which PCR was performed with the primer containing the adapter sequence (data not shown). The quality of the NGS data set can be assessed by ascertaining how high the coverage is, in terms of qualitative aspects: i) Tests 4 and 5 where PCR was performed with primers without fluorescent labeling and adapter sequences, and ii) Tests 2 and 3, in which the PCR was carried out with the included primers, and iii) Test 1 and 6, in which the PCR was performed with the fluorescently labeled primers, were found to be of high quality (data not shown). As a result, PCR products were obtained with primers that lacked the fluorescent label and adapter sequence under the above two conditions, and the best result was obtained when NGS was performed (Test 4, 5).

The lobSTR program or the Bowtie 2 program showed the best result in Test 4 and 5, especially when using the Bowtie 2 program, the same result as the CE when judging alleles based on a share of more than 20% As well as no allele in the range of 10% to less than 20%. In addition, when analyzed by lobSTR, alleles could not be determined in PentaD and VWA genetic loci, but Bowtie 2 was able to determine the allele correctly. The results from lobSTR showed an allele of +1 or 1 bp difference from the predicted allele size, but this phenomenon was not observed in Bowtie 2 using the reference sequence.

Meanwhile, the primer of the present invention applied degeneracy to the primer design so that even in the case of mutation of the base sequence of the primer, amplification occurs at the mutated binding site. The amplification product of the multiplex PCR using the primer of the present invention has a size of 20 bp - 600 bp, which is advantageous for amplification of a very small amount of template or degraded sample, thereby contributing to enhancement of detection sensitivity. According to one embodiment of the present invention, the size of the amplification product is 100 bp to 600 bp or 100 bp to 500 bp.

According to one embodiment of the present invention, the primer that binds complementarily to the D3S1358 genetic locus of step (a) of the present invention is a sequence of SEQ ID NOS: 1 and 2; The primer that binds complementarily to the TH01 genetic locus is a sequence of SEQ ID NO: 3 or SEQ ID NO: 4; The primers complementarily binding to the D21S11 genetic locus are those of SEQ ID NOS: 5 and 6; The primers complementarily binding to the D18S51 genetic locus are SEQ ID NOS: 7 and 8; Wherein the primer that binds complementarily to the PentaE gene locus is SEQ ID NOS: 9 and 10; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 11 and SEQ ID NO: 12; Wherein the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS 13 and 14; The primers that complementarily bind to the D7S820 genetic locus are those of Sequence Listing 15 and 16; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 17 and Sequence 18; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NOS: 19 and 20; Wherein the primers complementarily binding to the PentaD genetic locus are SEQ ID NOS 21 and 22, and the primers complementarily binding to the vWA genetic locus are SEQ ID NOS 23 and 24; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 25 and Sequence 26; Wherein the primer that binds complementarily to the TPOX genetic locus is SEQ ID NO: 27 and SEQ ID NO: 28; Wherein the primers complementarily binding to the FGA genetic locus are those of Sequence Listing Nos. 29 and 30; The primers complementarily binding to the amelogenin gene locus are SEQ ID NOS: 31 and 32.

The primer used in the gene detection method of the present invention has an optimal blending ratio for adjusting the peak balance.

According to one embodiment of the present invention, the primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.01-0.5 [mu] M, and the primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.01-0.4 [mu] M Wherein the primer complementarily binding to the D21S11 genetic locus has a final concentration of 0.4-0.8 μM and the primer complementarily binding to the D18S51 genetic locus has a final concentration of 0.3-0.7 μM and the PentaE genetic locus Has a final concentration of 1-1.4 [mu] M, and the primer that binds complementarily to the D5S818 genetic locus has a final concentration of 0.01-0.5 [mu] M, and the primer binding complementarily to the D13S317 genetic locus The primers have a final concentration of 0.2-0.6 [mu] M; The primer that binds complementarily to the D7S820 genetic locus has a final concentration of 0.1-0.5 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.2-0.6 [mu] M; The primer that binds complementarily to the CSF1PO genetic locus has a final concentration of 0.1-0.5 [mu] M; The primer that binds complementarily to the PentaD genetic locus has a final concentration of 1-1.4 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.01-0.4 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.3-0.7 [mu] M; The primer that binds complementarily to the TPOX genetic locus has a final concentration of 0.01-0.4 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.4-0.8 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.05-0.5 [mu] M. According to another embodiment of the invention, the primer complementarily binding to the D3S1358 genetic locus has a final concentration of 0.05-0.4 [mu] M and the primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.05-0.3 [mu] M Wherein the primer complementarily binding to the D21S11 genetic locus has a final concentration of 0.45-0.75 [mu] M, the primer complementarily binding to the D18S51 genetic locus has a final concentration of 0.35-0.65 [mu] M, and the PentaE gene locus Primers complementarily binding to the D5S818 genetic locus have a final concentration of 1.05-1.35 [mu] M, the primers complementarily binding to the D5S818 genetic locus have a final concentration of 0.05-0.4 [mu] M, and the complementary binding to the D13S317 genetic locus The primers have a final concentration of 0.25-0.55 [mu] M; The primer that binds complementarily to the D7S820 genetic locus has a final concentration of 0.15-0.45 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.25-0.55 [mu] M; The primer that binds complementarily to the CSF1PO gene locus has a final concentration of 0.15-0.45 [mu] M; The primer that binds complementarily to the PentaD gene locus has a final concentration of 1.05-1.35 [mu] M; The primers that complementarily bind to the vWA genetic locus have a final concentration of 0.05-0.35 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.35-0.65 [mu] M; The primer that binds complementarily to the TPOX genetic locus has a final concentration of 0.05-0.35 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.45-0.75 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.1-0.45 [mu] M. According to a particular embodiment of the invention, the primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.15-0.30 [mu] M, and the primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.05-0.2 [mu] M Wherein the primer complementarily binding to the D21S11 genetic locus has a final concentration of 0.5-0.7 [mu] M, the primer complementarily binding to the D18S51 genetic locus has a final concentration of 0.4-0.6 [mu] M, and the PentaE gene locus Primers complementarily binding to the D5S818 genetic locus have a final concentration of 1.1-1.3 [mu] M, and the primers complementarily binding to the D5S818 genetic locus have a final concentration of 0.15-0.3 [mu] M and are complementary to the D13S317 genetic locus The primers have a final concentration of 0.3-0.5 [mu] M; The primer that binds complementarily to the D7S820 genetic locus has a final concentration of 0.2-0.4 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.3-0.5 [mu] M; The primer that binds complementarily to the CSF1PO gene locus has a final concentration of 0.2-0.4 [mu] M; The primer that binds complementarily to the PentaD genetic locus has a final concentration of 1.1-1.3 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.1-0.25 μM; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.4-0.6 [mu] M; The primer that binds complementarily to the TPOX genetic locus has a final concentration of 0.1-0.25 μM; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.5-0.7 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.15-0.4 [mu] M.

The amelogenin gene locus is a genetic locus on a sex chromosome used to identify human sex. Amelogenin locus is identified as HUMAMELX when identifying the locus on the Y chromosome present in the male DNA, and as HUMAMELX when identifying the locus on the X chromosome present in the female DNA.

Step (b): The step of gene detection

Next, the allele genotype of the genetic locus is determined using the multiplex amplification product of step (a), and the human gene is identified as a gene.

According to one embodiment of the present invention, the nucleotide sequence of each STR allele amplified in the multiplex amplification product is compared with a reference sequence and evaluated, and as the coverage value in the STR allele, Determine the allelic genotype at STR locus.

The comparison / evaluation includes calculating a coverage value in each STR allele by aligning a nucleotide sequence of the STR allele with a reference sequence.

In the present invention, the reference sequence can be obtained by a method other than the lobSTR program (for example, (i) Illumina GAIIx system [3], Bowtie aligner [4], SAMtools [ (picard.sourceforge.net/), BEDtools [5], R program [6], and (ⅱ) 454 GS Junior System, CLC Genomic Workbench software. In the present invention, Bowtie 2 [8] is used which is based on the method used by Bornman et al. [3] and which has improved function than Bowtie.

In analyzing the STR with the lobSTR program, all the STR genomic residues in the human genome GRCh37 / hg19 are used as references. In the present invention, since only 15 STRs are analyzed, unnecessary STR genomic residues are reduced, A reference sequence was prepared and used so that the designation of the gene could be made easier.

The reference sequence was constructed by the following method:

The known number of repeats and their sequence to date in STR loci were obtained from STRbase [9]. The 5 'and 3' flanking region sequences of each STR were derived from the human genome GRCh37 / hg19, and the length of the peripheral sequences could be set to 500 bp - 550 bp. Basically, one STR consists of sequence of 5 'perimeter sequence, sequence of STR region, and sequence of 3' peripheral sequence. Based on this, a reference sequence for the STR genetic locus is constructed by sequentially linking the sequences from the smallest number of STR repeats to the greatest number sequence (see FIG. 1), as in the case of constructing a ladder of alleles in the CE system.

According to an embodiment of the present invention, the reference sequence includes a sequence of the STR region of the allele, a 5 'peripheral sequence of the STR region, and a 3' peripheral sequence of the STR region.

Since the reference sequence of the present invention is composed of fragments having various repetition times of STR (Short Tandem Repeat), it is used to evaluate correct alleles by analyzing the exact size and sequence of the multiplex amplification product. The reference sequence can be obtained not only by the method described above but also by a conventional method known in the art.

As used herein, the term ' STR ' refers to a tandem repeat sequence that is known to be widely distributed within an intron that does not contain genetic information such as a trait in the human genome, ), Which has been widely used worldwide. Many genetic loci in the human genome contain the STR region of the polymorphism. The STR locus consists of a short repeat sequence element, 2 to 7 base pairs in length, which is estimated to have 2 million trimer and tetramer STRs once every 15 kb in the human genome. As the number of short repeating units in a particular locus changes, the DNA length at that locus will vary with each allele and each individual. STR loci can be amplified by Polymerase Chain Reaction (PCR) using specific primer sequences identified on the side of this repeat sequence.

Alleles of these genetic loci are subdivided according to the number of copies of the repeated sequence contained within the amplified region, the repeat structure of alleles or sequence variation. That is, the analysis method of the present invention is capable of determining the repeating structure of STR alleles and observing the nucleotide sequence variation as NGS-based autosomal analysis method (Table 5 and Table 6), (i) (Ii) a different repeating structure between different samples in one genome, and (iii) the repeat structure of the STR region is the same, but a nucleotide sequence variation is observed in the peripheral region Can be observed. In these cases, nucleotide sequence-based analysis using NGS suggests that the STR allele identified by the existing CE can be further subdivided.

According to an embodiment of the present invention, in order to distinguish alleles of the same length, the number of copies of the repeated sequence (alleles) or sequence variation The identifying step may be additionally performed.

According to the present invention, a step of measuring the mixing ratio of the mixed sample using the sequence variation may be additionally performed. The mixing ratio is determined by analyzing the coverage ratio between alleles on each STR gene and analyzing the ratio of the reference base / mutation base of the sequence variation (see FIG. 3).

The multiplex amplification product of the present invention has a size of 20 bp to 600 bp. Small amplification products can be advantageous for amplification of very small template or degraded samples, resulting in improved detection sensitivity. According to an embodiment of the present invention, the multiplex amplification product is 100 bp to 600 bp or 100 bp to 500 bp (see FIGS. 2A to 2C).

The gene detection method of the present invention has the use of forensic typing or identification.

In addition, after step (b), a step of comparing the allele genotype of STR using the CE method can be additionally performed to determine the accuracy of the allelotype of STR determined by the method of the present invention.

(Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, vWA (human c-fms proto-oncogene for CSF-1 receptor gene), D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539 (NGS) -based primers, each containing a primer complementary to the von Willebrand factor A, D8S1179, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA) and amelogenin genetic loci -based mutant gene amplification kit for autosomal analysis.

Since the kit of the present invention utilizes the autosomal analysis method 1 of a human subject to be analyzed using the multiplex gene amplification described above, the common content between the two is to avoid the excessive complexity of the present invention, The description is omitted.

Ⅱ. NGS  base( next generation sequencing - based ) Autosomal  Analysis Method 2

According to another aspect of the present invention, the present invention provides a method of analyzing an autosomal analysis of an NGS-based next generation sequencing-based human subject, comprising the steps of:

(a) Human DnaSDNA, D5S818, Penta E, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), D7S820, D18S51, TPOX (human thyroid peroxidase gene), D16S539, D8S1179, Amplifier amplification was performed using primers complementary to the genetic locus of amelogenin, human fibrinogen alpha chain (FGA), D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA (von Willebrand factor A) ; And

(b) determining an STR (short tandem repeat) allele of the genetic locus using the NGS data of the multiplex amplification product of step (a), and identifying the human object as a gene.

The NGS method has been applied to the analysis of STR (short tandem repeat) markers and has been used primarily in the identification (identification) of human subjects in the forensic field. Although some studies have demonstrated the successful applicability of the NGS system to STR typing, there is no multiplex PCR system optimized for NGS analysis of forensic STR markers. Therefore, we constructed an in-house developed multiplex PCR system for the amplification of 18 markers (CODIS 13 STRs, D2S1338, D19S433, Penta D, Penta E and Ammelogenin) bp (more specifically 70 bp - 210 bp) size amplicons. PCR products were generated from single-source samples, mixed samples, and artificially resolved DNA samples using the multiplex PCR method of the present invention, and the resulting amplicon was used to prepare a barcoded library for sequencing in MiSeq Respectively. By performing NGS and analyzing its data, the inventors have confirmed that all STR genotype results are consistent with the results of CE-based typing. Sequence variations were also detected in the target STR region. In addition, when using the in-house developed multiplex PCR system, the STR loci that generated the large size amplicon in the commercial kit can effectively obtain the STR allele call and STR profile without loss of information, and the artificially degraded DNA To obtain STR profiles from NGS data. That is, the in-house developed multiplex PCR system of the present invention capable of generating small amplicons has been successfully used in forensic studies using the NGS system, STR NGS analysis using single-source samples, mixed samples and resolved DNA samples Can be applied.

Hereinafter, the gene detection method of the present invention will be described step by step in detail:

Step (a): Multiplex amplification step

First, the D19S433, D5S818, Penta E, CSF1PO (human c-fms proto-oncogene for CSF-1 receptor gene), D7S820, D18S51, TPOX (human thyroid peroxidase gene), D16S539, D8S1179, multiplex amplification is carried out using primers which bind to amelogenin, human fibrinogen alpha chain (FGA), D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA (von Willebrand factor A).

The DNA sample is a DNA sample isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or fetal cells, and mixtures thereof. And a biological sample. On the other hand, the DNA sample also includes a degraded DNA sample.

In the present invention, the multiplex amplification is a Polymerase Chain Reaction (PCR) amplification or a direct multiplex PCR amplification.

According to one embodiment of the present invention, the multiplex PCR amplification has an annealing temperature condition of 57-61 ° C. According to another embodiment of the present invention, the multiplex PCR amplification is performed at a temperature of 58-60 ° C Temperature conditions, and according to a particular embodiment of the invention, the multiplex PCR amplification has an annealing temperature condition of 59 < 0 > C.

The multiplex PCR amplification requires a reasonable number of cycles to perform PCR. According to one embodiment of the present invention, the multiplex PCR amplification is performed in 31-38 cycles, 31-35 cycles, 32-34 cycles, or 33 cycles. According to another embodiment of the present invention, when analyzing a degraded DNA sample, the multiplex gene Amplification is carried out in 34-38 cycles and in 36 cycles according to a particular embodiment of the invention.

The present invention is based on the finding that the DNA samples are complementary to the D19S433, D5S818, Penta E, CSF1PO, D7S820, D18S51, TPOX, D16S539, D8S1179, Amelogenin, FGA, D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA genetic loci Multiplex amplification is carried out using each of the primers that bind to each other.

According to one embodiment of the present invention, the primer used in the present invention is a non-labeled primer, which is a primer that is not labeled with a specific fluorescent dye or has no adapter.

Meanwhile, the primer of the present invention applied degeneracy to the primer design so that even in the case of mutation of the base sequence of the primer, amplification occurs at the mutated binding site. The amplification product of the multiplex PCR using the primer of the present invention has a size of 50 bp - 300 bp, which is advantageous for amplification of a very small amount of template or degraded sample, thereby contributing to improvement of detection sensitivity. According to one embodiment of the present invention, the size of the amplification product is 70 bp to 210 bp.

DNA sequence information for STR markers can be collected from the UCSC genome browser (http: // genome.ucsc.edu/). PCR primers for amplifying the respective markers basically satisfy the predetermined conditions on Primer3 (http://frodo.wi.mit.edu/primer3/input.htm) based on the collected nucleotide sequence information, Is designed to be 50 bp to 300 bp (see Table 7). The primer should be designed so that it is closest to the STR repeat unit in the range not overlapped with the STR repeat unit, and that there is no single nucleotide polymorphism (SNP) of 1% or more at the primer attachment site. In the case of multiple candidate primers designed with Primer3, the optimum primer combination without primer-to-primer interference in PCR amplification and the primer concentration maintaining the peak height balance between the STR genetic loci in electrophoresis were determined and multiplex PCR system Build.

According to one embodiment of the present invention, the primer that binds complementarily to the D19S433 genetic locus of step (a) is the sequence of SEQ ID NO: 33 and SEQ ID NO: 34; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 35 and SEQ ID NO: 36; Wherein the primer that binds complementarily to the Penta E gene locus is a sequence of SEQ ID NOS: 37 and 38; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NO: 39 and SEQ ID NO: 40; The primers complementarily binding to the D7S820 genetic locus are those of Sequence Listing 41 and Sequence 42; The primers complementarily binding to the D18S51 genetic locus are those of Sequence Listing 43 and Sequence 44; Wherein the primers complementarily binding to the TPOX genetic locus are those of Sequence Listing 45 and 46; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 47 and Sequence 48; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 49 and Sequence 50; Wherein the primers complementarily binding to the amelogenin gene locus are SEQ ID NO: 51 and SEQ ID NO: 52; Wherein the primers complementarily binding to the FGA gene locus are SEQ ID NOS: 53 and 54, and the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS: 55 and 56; The primers complementarily binding to the D2S1338 genetic locus are those of SEQ ID NOS: 57 and 58; The primers that complementarily bind to the D21S11 genetic locus are those of Sequence Listing 59 and Sequence 60; Wherein the primer complementarily binding to the Penta D genetic locus is a sequence selected from the group consisting of SEQ ID NOS: 61 and 62; The primers complementarily binding to the D3S1358 genetic locus are SEQ ID NO: 63 and SEQ ID NO: 64; Wherein the primers complementarily binding to the TH01 genetic locus are those of SEQ ID NOS: 65 and 66; The primers that complementarily bind to the vWA genetic locus are SEQ ID NO: 67 and SEQ ID NO: 68.

The primer used in the gene detection method of the present invention has an optimal blending ratio for adjusting the peak balance.

According to one embodiment of the invention, the primer complementarily binding to the D19S433 genetic locus has a final concentration of 0.4-0.8 [mu] M and the primer that binds complementarily to the D5S818 genetic locus has a final concentration of 0.3-0.8 [mu] M Wherein the primer complementarily binding to the Penta E genetic locus has a final concentration of 0.8-1.2 [mu] M, the primer complementarily binding to the CSF1PO genetic locus has a final concentration of 0.3-0.7 [mu] M, the D7S820 genetic The primers complementarily binding to the locus had a final concentration of 0.3-0.7 [mu] M, the primers complementarily binding to the D18S51 genetic locus had a final concentration of 0.7-1.1 [mu] M, and the TPOX genetic locus was complementarily bound Primers have a final concentration of 0.1-0.6 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.2-0.7 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.7-1.1 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.8-1.2 [mu] M; The primer that binds complementarily to the D13S317 genetic locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the D2S1338 genetic locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the D21S11 genetic locus has a final concentration of 0.5-0.9 [mu] M; The primer that binds complementarily to the Penta D genetic locus has a final concentration of 0.8-1.2 [mu] M; The primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.3-0.7 [mu] M; The primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.3-0.7 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.8-1.2 [mu] M. According to another embodiment of the present invention, the primer complementarily binding to the D19S433 genetic locus has a final concentration of 0.45-0.75 [mu] M and the primer that binds complementarily to the D5S818 genetic locus has a final concentration of 0.35-0.75 [mu] M Wherein the primer complementarily binding to the Penta E genetic locus has a final concentration of 0.85-1.15 [mu] M, the primer complementarily binding to the CSF1PO genetic locus has a final concentration of 0.35-0.65 [mu] M, the D7S820 genetic The primers complementarily binding to the locus had a final concentration of 0.35-0.65 [mu] M, the primers complementarily binding to the D18S51 genetic locus had a final concentration of 0.75-1.05 [mu] M, and the TPOX genetic locus was complementarily bound Primers have a final concentration of 0.15-0.55 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.25-0.65 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.75-1.05 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.65-0.95 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.85-1.15 [mu] M; The primer that binds complementarily to the D13S317 genetic locus has a final concentration of 0.65-0.95 [mu] M; The primer that binds complementarily to the D2S1338 genetic locus has a final concentration of 0.65-0.95 [mu] M; The primers complementarily binding to the D21S11 genetic locus had a final concentration of 0.55-0.85 [mu] M; The primer that binds complementarily to the Penta D gene locus has a final concentration of 0.85-1.15 [mu] M; The primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.35-0.65 [mu] M; The primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.35-0.65 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.85-1.15 [mu] M. According to a particular embodiment of the invention, the primer that binds complementarily to the D19S433 genetic locus has a final concentration of 0.5-0.7 [mu] M, and the primer that binds complementarily to the D5S818 genetic locus has a final concentration of 0.45-0.65 [mu] M Wherein the primer complementarily binding to the Penta E genetic locus has a final concentration of 0.9-1.1 [mu] M, the primer complementarily binding to the CSF1PO genetic locus has a final concentration of 0.4-0.6 [mu] M, the D7S820 genetic The primers complementarily binding to the locus had a final concentration of 0.4-0.6 [mu] M, the primers complementarily binding to the D18S51 genetic locus had a final concentration of 0.8-1 [mu] M, and the TPOX genetic locus was complementarily bound Primers have a final concentration of 0.25-0.45 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.35-0.55 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.8-1 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.7-0.9 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.9-1.1 [mu] M; The primer complementarily binding to the D13S317 genetic locus had a final concentration of 0.7-0.9 [mu] M; The primer that binds complementarily to the D2S1338 genetic locus has a final concentration of 0.7-0.9 [mu] M; The primer that binds complementarily to the D21S11 genetic locus has a final concentration of 0.6-0.8 [mu] M; The primer that binds complementarily to the Penta D gene locus has a final concentration of 0.9-1.1 [mu] M; The primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.4-0.6 [mu] M; The primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.4-0.6 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.9-1.1 [mu] M.

The amelogenin gene locus is a genetic locus on a sex chromosome used to identify human sex. Amelogenin locus is identified as HUMAMELX when identifying the locus on the Y chromosome present in the male DNA, and as HUMAMELX when identifying the locus on the X chromosome present in the female DNA.

Step (b): The step of gene detection

Next, the allele genotype of the genetic locus is determined using the multiplex amplification product of step (a), and the human gene is identified as a gene.

According to one embodiment of the present invention, the nucleotide sequence of each STR allele amplified in the multiplex amplification product is compared with a reference sequence and evaluated, and as the coverage value in the STR allele, Determine the allelic genotype at STR locus.

The comparison / evaluation includes calculating a coverage value in each STR allele by aligning a nucleotide sequence of the STR allele with a reference sequence.

In the present invention, a reference sequence is a reference sequence for efficiently analyzing a large amount of nucleotide sequence information generated in the NGS analysis. Reference sequences can be produced by the following method:

First, the number of repeats of the STR alleles known from the STRbase (http://www.cstl.nist.gov/biotech/strbase) to date, their sequence information, and the 5 'of each STR shown in the human genome GRCh37 / hg19 sequence 3 'flanking region sequence information. Based on these information, the length of the STR repeating unit peripheral sequence is designed to be 500 bp - 550 bp so that the NGS leads obtained through each primer combination can be aligned with the reference sequence. In addition, the STR 5 'peripheral sequence, the STR repeat sequence unit sequence, and the STR 3' peripheral sequence are combined for each allele using the macro function of Microsoft Excel.

According to an embodiment of the present invention, the reference sequence includes a sequence of the STR region of the allele, a 5 'peripheral sequence of the STR region, and a 3' peripheral sequence of the STR region, and the length of the peripheral sequence is 500 bp - Set to 550 bps.

NGS data analysis of acquired data can basically analyze NGS data using STRait Razor program, which is a program developed by Warshauer et al. [1] to determine STR alleles used mainly in the forensic science field. Bowtie 2 NGS data analysis can also be performed using program [2] and SAMtools [3], BEDtools [4], and Microsoft Excel. The coverage value for each STR allele genotype is calculated by the number of aligned leads having the sequence of the entire STR region according to the reference sequence information. Identification of repeat units based on nucleotide sequences for each STR allele can be done using the Integrative Genomics Viewer program [5, 6].

According to an embodiment of the present invention, a single-source sample or a mixture thereof (for example, a mixed sample mixed at a ratio of 1: 1, 1: 3, 1: 6, 1: 9) , A new multiplex PCR system of the present invention can be used to generate amplification products and sequence information can be obtained using an appropriate sequencing system (Roche's GS FLX or Illumina's MiSeq platform).

The method of the present invention includes the step of identifying the number of copies of the repeat sequence, repeat structure of alleles or sequence variation in the STR genomic locus to distinguish alleles of the same length Can be additionally performed.

According to the present invention, a step of measuring the mixing ratio of the mixed sample using the sequence variation may be additionally performed. The mixing ratio is determined by analyzing the coverage ratio between alleles on each STR gene and analyzing the base / mutation base ratio of the sequence mutation.

The autosomal analysis method of the present invention has the use of forensic typing or identification.

In addition, after step (b), a step of comparing the allele genotype of STR using the CE method can be additionally performed to determine the accuracy of the allelotype of STR determined by the method of the present invention. That is, a single sample and a mixed sample are subjected to PCR using a commercial kit (e.g., AmpFlSTR Identifiler (Applied Biosystems)) according to the manufacturer's instructions, and then analyzed using an analysis system (e.g., ABI PRISM3130xl Genetic Analyzer (Applied Biosystems) ]. Next, the STR allele genotype determined in a program such as ABI GeneMapperID software version 3.7 (Applied Biosystems) is compared with the results of the STR allelic genotype obtained by the NGS method.

According to another aspect of the present invention, there is provided a method for screening for a human thyroid peroxidase gene (D19S433, D5S818, Penta E, CSF1PO (human c-fms proto-oncogene for CSF-1 receptor gene), D7S820, D18S51, TPOX NGS-based primers containing respective primers complementary to the genetic locus of amelogenin, human fibrinogen alpha chain (FGA), D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA (von Willebrand factor A) (next generation sequencing-based) autosomal analysis multiplex gene amplification kit.

Since the kit of the present invention utilizes the autosomal analysis method 2 of the human subject to be analyzed using the multiplex gene amplification described above, the common content between the two is to avoid the excessive complexity of the present invention, The description is omitted.

The features and advantages of the present invention are summarized as follows:

(a) The present invention provides an autosomal analysis method of a next generation sequencing-based human subject and a multiplex PCR system optimized therefor.

(b) By analyzing NGS data of the STR marker using the method of the present invention, it is possible to accurately and effectively analyze the autosomal chromosome of a human subject by analyzing STR allelotype determination, repetitive structure of alleles, and nucleotide sequence variation can do.

(c) The present invention provides a size optimized multiplex PCR system for NGS analysis capable of producing amplicons of 50 bp to 300 bp in size.

(d) The method of the present invention using the NGS system can be useful for analyzing and interpreting STR profiles in single-source samples, mixed samples, and degraded DNA samples in forensic studies.

1 shows a schematic diagram of an STR reference sequence. Long flanking sequences ranging from 500 bp to 550 bp in the STR reference tm sequence were designed to be completely aligned with the sample sequence generated by the specific primer combination.
Figures 2a-2c illustrate the quality verification of a library constructed on a high sensitivity chip using a 2100 Bioanalyzer. Sections smaller than 100 bp containing the adapter dimer were successfully removed. a: Standard male DNA 2800M; b: Standard female DNA 9947A; c: 1: 1 mixture.
Figures 3A-3C show the reference / mutation rate-based mixing ratio measurements from sequence variations observed in D13S317 locus. Sequence variation from adenine (A) to thymine (T) was observed at the 3 'flanking site of D13S317 locus. The mixing ratio was measured as 46% (A): 53% (T). 3a: Standard male DNA 2800M; 3b: Standard female DNA 9947A; 3c: 1: 1 mixture; 3d: mixing ratio.
Figure 4 shows the 18 markers of an in-house developed multiplex PCR system for NGS analysis.
FIG. 5 shows a coverage histogram of each allele from MiSeq data. The Y axis represents the total coverage percentage, and the number at the top represents the number of reads of each marker.
Figure 6 shows the electrophorogram of the amplicon produced by multiplex PCR. a 2800M and b 9947A control DNA.
Figures 7a-7e show the nucleotide sequence variations observed in two control sample DNA samples (a: D2S1338, b: D3S1358, c: D8S1179, d: D21S11, e: vWA). The primer sequences and flanking regions are not shown in the figure. Blue letters = core repeat unit, orange letters = repeat units, pink letters = non-repeat region, bold red letters = sequence variation.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by 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

Example 1: Sequence Generation and Genotyping of 15 Stage Chromosomal Strains Using Next Generation Sequence Analysis

end. Research method

1. DNA sample

The DNA samples used were a commercial male standard sample 2800M (Promega, Madison, Wis., USA), which was used as a control in the genetics research of the law, and a female 9947A (Promega). These DNA samples were quantified using a NanoDrop 1000 spectrophotometer (Thermo. Fisher Scientific, Waltham, Mass., USA) and then prepared at a concentration of 1 ng / μl. The 1: 1 mixed sample was mixed with two single samples (2800M and 9947A) to a final concentration of 1 ng / μl.

2. Generation and identification of STR amplification products

In order to analyze 15 STR genomes and amelogenins of D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO, PentaD, vWA, D8S1179, TPOX and FGA, the PowerPlex 16 system ) Was prepared based on the disclosed information of the primers. However, unlike the conventional STR method using CE, no fluorescent marker was attached to the primer. Table 1 shows the sequence and final concentration of the primers used in the PCR procedure. PCR was carried out in a total volume of 25 μl containing 2.5 μl of 10 × Gold ST * R buffer (Promega), 4.0 units of AmpliTaq Gold DNA polymerase (Applied Biosystems, Foster City, CA, USA), 1 ng of primer and 1 ng of DNA sample Was prepared by adjusting the PCR temperature cycle to 34 cycles in the method recommended by the PowerPlex 16 system. After completion of the PCR, polyacrylamide gel ST * R bufforesis was used to confirm that the amplified products were uniformly generated. 2.5 Amplification products were purified using QIAquick PCR purification kit (QIAGEN, Hilden, Germany). The concentration of the obtained amplification product was measured using a QuantiT ^™ PicoGreen dsDNA Assay Kit (Invitrogen, Carlsbad, Calif., USA), and the purity was measured at 260 nm and 280 nm with a NanoDrop 1000 spectrophotometer By calculating the ratio of absorbance.

Primer set of multiplex PCR system ^* Final concentration - Locus Primer set The primer sequence (5` → 3`) Concentration (μM) One D3S1358 D3-PP16-F ACTGCAGTCCAATCTGGGT 0.20 D3-PP16-R ATGAAATCAACAGAGGCTTGC 2 TH01 TH01-PP16-F GTGATTCCCATTGGCCTGTTC 0.10 TH01-PP16-R ATTCCTGTGGGCTGAAAAGCTC 3 D21S11 D21-PP16-F ATATGTGAGTCAATTCCCCAAG 0.60 D21-PP16-R TGTATTAGTCAATGTTCTCCAGAGAC 4 D18S51 D18-PP16-F TTCTTGAGCCCAGAAGGTTA 0.50 D18-PP16-R ATTCTACCAGCAACAACACAAATAAAC 5 Penta E PentaE-PP16-F ATTACCAACATGAAAGGGTACCAATA 1.20 PentaE-PP16-R TGGGTTATTAATTGAGAAAACTCCTTACAATTT 6 D5S818 D5-PP16-F GGTGATTTTCCTCTTTGGTATCC 0.20 D5-PP16-R AGCCACAGTTTACAACATTTGTATCT 7 D13S317 D13-PP16-F ATTACAGAAGTCTGGGATGTGGAGGA 0.40 D13-PP16-R GGCAGCCCAAAAAGACAGA 8 D7S820 D7-PP16-F ATGTTGGTCAGGCTGACTATG 0.30 D7-PP16-R GATTCCACATTTATCCTCATTGAC 9 D16S539 D16-PP16-F GGGGGTCTAAGAGCTTGTAAAAAG 0.40 D16-PP16-R GTTTGTGTGTGCATCTGTAAGCATGTATC 10 CSF1PO CSF1PO-PP16-F CCGGAGGTAAAGGTGTCTTAAAGT 0.30 CSF1PO-PP16-R ATTTCCTGTGTCAGACCCTGTT 11 Penta D PentaD-PP16-F GAAGGTCGAAGCTGAAGTG 1.20 PentaD-PP16-R ATTAGAATTCTTTAATCTGGACACAAG 12 Amelogenin Amelo-PP16-F CCCTGGGCTCTGTAAAGAA 0.25 Amelo-PP16-R ATCAGAGCTTAAACTGGGAAGCTG 13 vWA vWA-PP16-F GCCCTAGTGGATGATAAGAATAATCAGTATGTG 0.15 vWA-PP16-R GGACAGATGATAAATACATAGGATGGATGG 14 D8S1179 D8-PP16-F ATTGCAACTTATATGTATTTTTGTATTTCATG 0.50 D8-PP16-R ACCAAATTGTGTTCATGAGTATAGTTTC 15 TPOX TPOX-PP16-F GCACAGAACAGGCACTTAGG 0.15 TPOX-PP16-R CGCTCAAACGTGAGGTTG 16 FGA FGA-PP16-F GGCTGCAGGGCATAACATTA 0.60 FGA-PP16-R ATTCTATGACTTTGCGCTTCAGGA

* Each primer sequence based on information from the PowerPlex 16 system without fluorescent dye

3. Library using amplification product ( library ) Production

The first step for NGS analysis was to use the GS Rapid Library Preparation Kit (Roche Diagnostics Corp., Branford, CT, USA) to construct a library to attach a specific adapter to the amplification product Respectively. In this process, the adapter containing the Multiplex Identifier (MID) was used to distinguish the DNA samples. AMPure bead (Beckman Coulter, Brea, CA, USA) was used to purify the library so that the ratio of the amplified product to the bead was 2: 1 so that small fragments smaller than 100 bp could be removed. Respectively. The size distribution and the concentration of the final library were checked using a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA).

4. Clone amplification clonal amplification ) And large base sequence generation

Emulsion PCR for clone amplification of the prepared library was performed using the GS Junior Titanium emPCR Kit (Lib-L; Roche Diagnostics Corp.) according to the manufacturer's instructions. For this, the library was diluted to 1 × 10 ⁷ molecules per μl after calculating the number of molecules per μl based on the measured concentration (Eq. 1). Also Scheible such as ¹⁴⁾ can copy per bead As presented was set to 1.0. Generation of large base sequences for the emulsion PCR amplification products was performed on GS Junior (Roche Diagnostics Corp.) equipment using the GS Junior Titanium Sequencing Kit (Roche Diagnostics Corp.) according to the manufacturer's instructions.

: Eq. 1
Molecules / μl =

: Eq. One

5. NGS  Analysis of data

Based on the protocol proposed by Bornman et al. (15) for STR allelic genotyping, i) production of a reference sequence, ii) alignment between NGS leads and reference sequences, iii) coverage of each STR allele And iv) the order of allele genotyping in each STR genome was analyzed. The number of repetitions of STR alleles known to date for the construction of reference sequences and their sequences were obtained from STRbase (http://www.cstl.nist.gov/biotech/strbase) and the 5 'and 3' The flanking region sequence was derived from the human genome GRCh37 / hg19. In addition, by setting the length of the peripheral sequence to 500-550 bp, alignment of the NGS leads obtained through any combination of primers with the reference sequence was made possible (FIG. 1). Finally, the reference sequence was constructed using the macro function of Microsoft Excel so that it could consist of the 5 'perimeter sequence, the STR region sequence, and the 3' perimeter sequence.

The alignment between the NGS lead and the reference sequence was done using the Bowtie 2 ¹⁶⁾ program on the Linux operating system. SAMtools ^{17 )} and BEDTool ^{18 )} were used in order to convert the format of the obtained result file. The coverage value for each STR allele was obtained by calculating the number of leads containing the entire STR region among the leads aligned in the reference sequence. For the determination of allelic genotypes in each STR genotype, the reference value was applied to 20% of the total coverage value in each genome and 10% in the mixed sample in a single sample. Identification of the repeat structure based on the nucleotide sequence of each STR allele determined above was performed using the Integrative Genomics Viewer ^{19 )} . In addition, a method of examining the ratio of the coverage value to the allelic genotype in each STR genome, and identifying the sequence variation at a specific site, Respectively.

6. Capillary electrophoresis ( capillary 전공기 ) STR  analysis

To determine whether the allele genotypes of male and female standard and 1: 1 mixed samples from NGS data were correctly determined, allelic types of these samples were examined by capillary electrophoresis based STR analysis. PCR was performed using 1 ng of each DNA sample and PowerPlex 16 HS system (Promega) according to the manufacturer's instructions. The obtained amplified products were detected using ABI PRISM 3130xl Genetic Analyzer and GeneScan Software Version 3.7 (Applied Biosystems) Finally, analysis was performed using GeneMapper ™ ID Software Version 3.1 (Applied Biosystems).

Results

One. PCR  Production of libraries from amplification products

PCR was performed from 2800M standard male DNA samples, 9947A standard female DNA samples, and 1: 1 mixture samples using primers prepared based on the Powerplex 16 system information. After the obtained amplification products were purified, their purity was measured. At this time, amplification products were obtained in the range of 1.92-1.95 and in the range of 1650-2220 ng. Therefore, the libraries were constructed from the amplification products of three DNA samples, since the minimum amount of 500 ng or more and the purity of 1.70 or more and less than 2.00 were met for the library production proposed by Roche. FIGS. 2A to 2C show the sizes of the libraries of the final samples obtained through the Bioanalyzer. Since small fragments of less than 100 bp are rarely observed, it was confirmed that small fragments were selectively removed by the bead method in the library production process.

2. NGS  Of the material By sample  Sort and sequence alignment ( sequence alignment )

The total number of leads obtained from NGS was 164,468, and their average length was 183.64 bp. According to the classification using the MID sequence, 51,475 standard specimens of 2800M, 33,213 standard specimens of 9947A, and 1 of them : 76,943 mixed samples and 2,837 untested leads were obtained. By aligning these data with reference sequences, we could confirm the number of leads obtained from each STR genome (Table 2). In the 15 STR genotypes D3S1358, D5S818, D13S317 and TH01, more lead counts were obtained than other genome sequences. In the case of D16S439, D18S51, CSF1PO, FGA, Penta D, Penta E and TPOX, relatively small leads were obtained, and the size of these amplified products was generally larger than 250 bp. When the percentage of the number of leads including the entire STR region with respect to the number of all leads in each dielectric layer was examined, it was found that D18S51, FGA, Penta D and Penta E showed less than 50%. Likewise, the larger the size of the amplification product, the smaller the number of leads containing the entire STR region.

The read counts of 15 STR loci in each sample STR locus Amplicon Size Range (bp) 2800M 9947A 1: 1 mixture All * Full STR ¹ Total STR / All (%) All * Full STR ¹ Total STR / All (%) All * Full STR ¹ Total STR / All (%) D3S1358 115-147 9470 8743 92.3 6341 6012 94.8 14261 13306 93.3 D5S818 119-155 9485 8705 91.8 5523 5011 90.7 9347 8531 91.3 D7S820 215-247 3676 3476 94.6 1868 1780 95.3 4815 4603 95.6 D8S1179 203-247 4458 4017 90.1 1967 1805 91.8 3368 3054 90.7 D13S317 169-201 4897 4631 94.6 4060 3868 95.3 12839 12140 94.6 D16S539 264-304 967 877 90.7 708 655 92.5 2497 2361 94.6 D18S51 209-366 739 332 44.9 1284 546 42.5 1117 481 43.1 D21S11 203-259 3045 2313 76.0 2996 2525 84.3 4873 3871 79.4 CSF1PO 321-357 291 244 83.8 596 522 87.6 862 742 86.1 FGA 322-444 956 460 48.1 666 255 38.3 3137 1440 45.9 Penta D 376-441 142 31 21.8 267 56 21.0 403 75 18.6 Penta E 379-474 193 84 43.5 356 116 32.6 563 309 54.9 TH01 156-195 5503 4620 84.0 3324 2811 84.6 6712 5518 82.2 TPOX 262-290 269 230 85.5 215 183 85.1 679 576 84.8 vWA 123-171 3153 2782 88.2 1014 919 90.6 8565 7649 89.3 AMEL 106, 112 3416 3247 95.1 1773 1741 98.2 2334 2247 96.3 Sum - 50550 44792 88.6 32958 28805 87.4 76372 66903 87.6

* Lead All aligned leads, regardless of STR presence or absence

† Aligned leads that contain the entire STR portion, and all STRs that are less than 50% are shown in bold

3. STR  Determination of repetitive structure of alleles and Nucleotide sequence variation  Confirm

Table 3 shows an example in which STR alleles are determined from NGS data for two single samples (2800M and 9947A) and a 1: 1 mixture thereof. In this way, alleles at 15 STR loci were determined for these samples (Table 4). In order to confirm whether the genotypes determined from the NGS data match exactly, we compared the STR genotypes determined by the conventional CE method. The results were consistent with all 15 STR genotypes in a single sample, and 13 STR genotypes in a 1: The alleles were exactly identical in the left (D3S1358, D5S818, D7S820, D8S1179, D13S317, D16S539, D18S51, D21S11, CSF1PO, FGA, Penta D, TH01 and TPOX) I did. Based on their coverage values, the Penta E allele 12 and vWA allele 12 were 7.44% and 9.11%, respectively, which was below the reference value of 10% of the mixed sample.

Identification of the D3S1358 allele based on the allele coverage percentage in single-source and 1: 1 mixtures Allele 2800M 9947A 1: 1 mixture Allele lead count Allele coverage * (%) Allele lead count Allele coverage * (%) Allele lead count Allele coverage * (%) 11 0 - 2 0.03 0 - 12 0 - 12 0.20 5 0.04 13 2 0.02 217 3.61 103 0.77 14 13 0.15 2868 47.70 1519 11.41 15 71 0.81 2879 47.89 2245 16.87 16 495 5.66 34 0.57 541 4.06 17 4355 49.81 0 - 4936 37.09 18 3757 42.97 0 - 3906 29.35 19 24 0.27 0 - 33 0.25 20 26 0.30 0 - 21 0.16 Sum 8743 - 6012 - 13309 -

* The shaded area (bold) in the above table represents the allele based on the analysis limits.

* Allele coverage percentage (%) = allele lead count / locus lead count x 100

STR Genotyping results in 2 single-source and 1: 1 mixtures by CE and NGS assays STR locus 2800M 9947A 1: 1 mixture CE NGS CE NGS CE NGS D3S1358 17, 18 17, 18 14, 15 14, 15 14, 15, 17, 18 14, 15, 17, 18 D5S818 12 12 11 11 11, 12 11, 12 D7S820 8, 11 8, 11 10, 11 10, 11 8, 10, 11 8, 10, 11 D8S1179 14, 15 14, 15 13 13 13, 14, 15 13, 14, 15 D13S317 9, 11 9, 11 11 9, 11 9, 11 9, 11 D16S539 9, 13 9, 13 11, 12 11, 12 9, 11, 12, 13 9, 11, 12, 13 D18S51 16, 18 16, 18 15, 19 15, 19 15, 16, 18, 19 15, 16, 18, 19 D21S11 29, 31.2 29, 31.2 30 30 29, 30, 31.2 29, 30, 31.2 CSF1PO 12 12 10, 12 10, 12 10, 12 10, 12 FGA 20, 23 20, 23 23, 24 23, 24 20, 23, 24 20, 23, 24 Penta D 12, 13 12, 13 12 12 12, 13 12, 13 Penta E 7, 14 7, 14 12, 13 12, 13 7, 12, 13, 14 7, (12), 13, 14 TH01 6, 9.3 6, 9.3 8, 9.3 8, 9.3 6, 8, 9.3 6, 8, 9.3 TPOX 11 11 8 8 8, 11 8, 11 vWA 16, 19 16, 19 17, 18 17, 18 16, 17, 18, 19 16, 17, (18), 19

* Parentheses: Alleles with a value lower than 10% of the total coverage value (true allele)

From the NGS data of male and female standard DNA samples, the nucleotide sequence of the alleles determined at the 15 STR loci was confirmed and based on this, it was possible to determine the repeat structure of each STR region (Table 5). In addition, the nucleotide sequences of the STR genomic loci were compared among the samples, and the differences in the repeating structure or the sequence of the nucleotide sequences were observed as follows. The first is that the two alleles have the same length but different base sequences. The D8S1179 genetic locus of the 9947A sample appears homozygous with allelic variants of 13 and 13 in the CE-based method. One result of analysis by NGS is "TCTA TCTG [TCTA] ₁₁ " and the other is " And [TCTA] ₁₃ ", respectively. As a result, the STR region is heterozygous, having a different base sequence but having a length. The second case is that the samples have different repeating structures. In the D3S1358 genetic locus, the core repeat unit is [TCTA], and repeat units of [TCTG] are found, depending on the sample. Comparing the repeating structures of D3S1358 genetic loci between 2800M and 9947A samples, it was observed that the positions of [TCTG] appeared to be different from each other in the third and the second. Third, there is a case in which nucleotide sequence variation is observed in a peripheral region other than an STR region. The STR allelic genotype was the only one found in the D13S317 genetic locus of the 2800M sample identified as 9, 11. Alleles 9 in the 3 'perimeter sequence were identified as "AATC AATC" and allele 11 as "TATC AATC". It was observed as if one more iteration of [TATC] was added.

Repetition structure of 15 STRs in Two Standard Sample from NGS data -2800M STR locus Zino type Core iteration Repeating structure D3S1358 17, 18 TCTA 17: TCTA [TCTG] ₃ [TCTA] ₁₃ 18: TCTA [TCTG] ₃ [TCTA] ₁₄ D5S818 12 AGAT 12: [AGAT] ₁₂ D7S820 8, 11 GATA 8: [GATA] ₈ 11: [GATA] ₁₁ D8S1179 14, 15 TCTA 14: TCTA TCTG [TCTA] ₁₂ 15: [TCTA] ₂ TCTG [TCTA] ₁₂ D13S317 9, 11 TATC 9: [TATC] ₉ [ AATC ] ₂ 11: [TATC] ₁₁ TATC AATC D16S539 9, 13 GATA 9: [GATA] ₉ 13: [GATA] ₁₃ D18S51 16, 18 AGAA 16: [AGAA] ₁₆ AAAG [ AG ] ₃ 18: [AGAA] ₁₈ AAAG [ AG ] ₃ D21S11 29, 31.2 TCTA 29: [TCTA] ₄ [TCTG] ₆ [TCTA] ₃ TA [TCTA] ₃ TCA [TCTA] ₂ TCCA TA [TCTA] ₁₁ 31.2: [TCTA] ₅ [TCTG] ₆ [TCTA] ₃ TA [TCTA] ₃ TCA [TCTA] ₂ TCCA TA [TCTA] ₁₁ TA TCTA CSF1PO 12 AGAT 12: [AGAT] ₁₂ FGA 20, 23 CTTT 20: [TTTC] ₃ TTTT TTCT [CTTT] ₁₂ CTCC [TTCC] ₂ 23: [TTTC] ₃ TTTT TTCT [CTTT] ₁₅ CTCC [TTCC] ₂ Penta D 12, 13 AAAGA 12: [AAAGA] ₁₂ 13: [AAAGA] ₁₃ Penta E 7, 14 AAAGA 7: [AAAGA] ₇ 14: [AAAGA] ₁₄ TH01 6, 9.3 AATG 6: [AATG] ₆ 9.3: [AATG] ₆ ATG [AATG] ₃ TPOX 11 AATG 11: [AATG] ₁₁ vWA 16, 19 TCTA 16: TCTA [TCTG] ₃ [TCTA] ₁₂ TCCA TCTA 19: TCTA [TCTG] ₄ [TCTA] ₁₄ TCCA TCTA

Repeating structure of 15 STRs in Two Standard Sample from NGS data -9947AM STR locus Zino type Core iteration Repeating structure D3S1358 14, 15 TCTA 14: TCTA [TCTG] ₂ [TCTA] ₁₁ 15: TCTA [TCTG] ₂ [TCTA] ₁₂ D5S818 11 AGAT 12: [AGAT] ₁₁ D7S820 10, 11 GATA 10: [GATA] ₁₀ 11: [GATA] ₁₁ D8S1179 13 TCTA 13a: TCTA TCTG [TCTA] ₁₁ 13b: [TCTA] ₁₃ D13S317 11 TATC 11: [TATC] ₁₁ [ AATC ] ₂ D16S539 11, 12 GATA 11: [GATA] ₁₁ 12: [GATA] ₁₂ D18S51 15, 19 AGAA 15: [AGAA] ₁₅ AAAG [ AG ] ₃ 19: [AGAA] ₁₉ AAAG [ AG ] ₃ D21S11 30 TCTA 30: [TCTA] ₆ [TCTG] ₅ [TCTA] ₃ TA [TCTA] ₃ TCA [TCTA] ₂ TCCA TA [TCTA] ₁₁ CSF1PO 10, 12 AGAT 10: [AGAT] ₁₀ 12: [AGAT] ₁₂ FGA 23, 24 CTTT 23: [TTTC] ₃ TTTT TTCT [CTTT] ₁₅ CTCC [TTCC] ₂ 24: [TTTC] ₃ TTTT TTCT [CTTT] ₁₆ CTCC [TTCC] ₂ Penta D 12 AAAGA 12: [AAAGA] ₁₂ Penta E 12, 13 AAAGA 12: [AAAGA] ₁₂ 13: [AAAGA] ₁₃ TH01 8, 9.3 AATG 8: [AATG] ₈ 9.3: [AATG] ₆ ATG [AATG] ₃ TPOX 8 AATG 8: [AATG] ₈ vWA 17, 18 TCTA 17: TCTA [TCTG] ₄ [TCTA] ₁₂ TCCA TCTA 18: TCTA [TCTG] ₄ [TCTA] ₁₃ TCCA TCTA

4. Estimation of Mixing Ratio in Mixed Samples

The ratio of 1: 1 mixed sample was estimated by examining the ratio of coverage values to alleles in each of the 15 STR genotypes analyzed, and identifying sequence variations at specific positions and determining the ratio of each base. The allele genotype for the 1: 1 mixture of these genes is "14, 15, 17, 18" because the D3S1358 genotype has the allele of 17, 18 in 2800M and 14, 15 in 9947A. . Theoretically, the coverage ratio for each allele genotype was expected to be 1: 1: 1: 1, but their coverage values were in the order of 1519, 2245, 4936, and 3006 (Table 3) : 1.5: 3.3: 2.6 ". In addition, in the D13S317 genetic locus of the 2800M sample, nucleotide sequence change from adenine to thymine was confirmed based on the human reference genome hg19 in the 3 'peripheral sequence of the allele 11 (FIG. 3). As a result of investigation of the number of each base at this position, thymine was found to be 3037 (46%) and adenine was found to be 2642 (53%) among the total coverage value of 5683.

However, considering that the allele genotype at D13S317 in the 2800M sample is 9 and 11 heterozygotes and 9947A is homozygous for 11 and 11, the ratio of adenine to thymine should be 2: 1. As a result, it was found that the actual mixing ratio was different from the expected mixing ratio.

All. Review

Analysis of STR genotypes using the NGS method consists of the production of STR amplification products, library production, mass sequencing, and data analysis. In this study, we constructed a multiplex amplification PCR system using the primer information of PowerPlex 16 system used for CE-based autosomal STR analysis in the forensic field, and generated the amplification product of the standard DNA sample and analyzed the result obtained through NGS Respectively. This study is similar to the NGS method for forensic STRs reported in several groups. ^{10-15) In} the first study of Van Neste et al. ( ^{10 )} , amplification products for single and mixed samples were prepared and NGS analysis was performed using a commercially available STR kit capable of analyzing 9 STR genome loci. Here, we used primers with fluorescent markers as they were, and the authors confirmed that NGS analysis showed a large difference in the number of leads read in both forward and reverse directions, and this was presumed to be due to fluorescent markers . In this study, as in the second study of Van Neste et al. ( ^{20 )} , amplification products were prepared by multiplex amplification PCR with primers without fluorescent markers attached to 15 STR genomic loci. However, other NGS equipment, GS Junior .

Roche recommends the use of i) a primer (fusion primer) in which an adapter sequence and a template specific sequence are bound to each other to produce an amplification product, or ii) It is a method of attaching the adapter after performing a process (fragmentation) of making the DNA into a small intercept. First, the fusion primer method has been used to perform amplification product generation and library production at the same time. However, the amplification product was not generally obtained, and thus the total number of leads obtained by NGS was small, and some alleles were not correctly determined in some genetic loci. Perhaps because of the use of longer primers, the efficiency of amplification in the PCR process was reduced, and it was also thought to have influenced the subsequent emulsion PCR steps and was not used in this study. The second method of fragmenting DNA is to construct a library when the whole genome and mitochondrial DNA are long, and then construct amplification products in the range of 100-450 bp by multiplex amplification PCR. It was not appropriate for analysis. However, this amplification product was regarded as a small section that had already been segmented, and NGS data could be successfully generated through library production by attaching the adapter. As a result, the method of constructing the above-mentioned library while maintaining the conventional multi-amplification PCR method will be very useful for the study of STR genetic loci through NGS in the forensic science field. NGS data were generated for 15 STR genomic loci, and when the distribution of leads for each genome locus was analyzed by analysis, it was observed not to appear constant but to be inversely proportional to the size of the amplified product (Table 2). Generally, the number of leads was large in the genome where the amplification product was made smaller than that based on 250 bp, but the number of leads was relatively small for the genome left large. Especially, in D18S51, FGA, Penta D, and Penta E in which amplification products of 300 bp or more are generated, not only the number of all leads was obtained, but also the ratio of the leads including the entire STR region (Entire STR / All) %. In this study, because the primer information of PowerPlex 16 system was used for NGS, the size of amplification product is also widely ranged from 106 to 474 bp. Considering these points, it is expected that, if the amplification products are reduced in size and narrowed to produce them, the number of leads in each STR genome will be increased, thereby providing confidence in the results of the subsequent analysis. Therefore, a new experimental design is needed to obtain STR analysis results optimized for NGS. It is also possible to read through the performance enhancements of the MiSeq (Illumina Inc., San Diego, CA, USA) and Ion Torrent PGM (Life Technologies, Carlsbad, CA, USA) As the length of the leads is getting longer, a design that can be applied together in these devices will be required.

Compared with the result obtained by the CE method after determining STR alleles by NGS, single samples and 1: 1 mixed samples showed allele genotypes in all STR genotypes in a single sample, whereas some of the 1: 1 mixed samples In the STR genotype (PentaE, vWA), it was confirmed that the allelic genotype obtained by the CE method and the allele genotype obtained by the NGS analysis do not match (Table 4). This is because the coverage value of each single allele in these genetic loci is less than the reference value (10%) set for the allele determination in this study. However, these alleles were larger than the allergic coverage values considered to be stuttering, so it was not appropriate to exclude them from the results. In the future, it will be necessary to take careful efforts to ensure that there is no error in the analysis results in consideration of the above points when determining alleles in STR analysis of mixed samples using NGS.

It was possible to determine the repeat structure of STR alleles and to observe the nucleotide sequence variation by the NGS technique (Table 5), and three characteristics were confirmed in two male and female standard samples (2800M and 9947A). The first one was the same length of alleles in one genome, but the other had the same sequence. The second one showed a different repetition structure among the different samples in one genome. Were the same, but the nucleotide sequence variation was observed in the peripheral part. These findings suggest that the STR allele identified through CE can be further subdivided by nucleotide sequence analysis using NGS. In addition, as shown in the previous study ^{11 , if} nucleotide sequence information of STR allele using NGS and their frequency data are constructed in Korean, it will be useful for forensic science practice such as paternity identification and crime investigation.

It has been confirmed that the ratio of the coverage value to the STR allele in the 1: 1 mixed sample is different from the expected ratio in estimating the mixing ratio by NGS analysis. In particular, when the alleles derived from 2800M and 9947A were isolated and their coverage values were examined, it was found that the alleles from 2800M and 9947A did not show the same pattern but shifted toward the 2800M alleles (Table 3). This pattern was the same for all 15 STR strains (data not shown). In addition, it was observed that the number of adenine and thymine from the nucleotide sequence variation observed in the D13S317 genetic locus was estimated to be higher than expected at 2800M (FIG. 3) even when the mixing ratio was estimated. To investigate the cause, we investigated the peak height of the allele in the profile of the 1: 1 mixed sample obtained through CE and estimated the mixing ratio. CE results showed that the alleles of one sample were more than expected as well as NGS results (data not shown). CE and NGS techniques commonly begin by preparing amplification products from PCR from the target sample. This suggests that the above phenomenon is due to the difference in amplification efficiency between the two samples generated in the PCR process. Therefore, when estimating the mixing ratio using NGS, this point should be fully considered and analyzed.

In this study and Bornman et al. [ ^{15 ]} , two single samples were used for analysis of "1: 1" through NGS. This case can be applied to the interpretation of the samples obtained at the case site consisting of one suspect and one victim. However, if one of the samples shows a low ratio, the interpretation of the data may become difficult. Therefore, in order to take into account the actual characteristics of the sample obtained at the site of the accident, it is necessary to investigate whether effective data interpretation is possible for the mixed samples in a more varied ratio besides the "1: 1" condition.

Van Neste et al. Prepared a mixed sample from four samples at a ratio of "10: 20: 30: 40" and "93.40: 5: 1: 0.5: ^{20) Since} the minimum standard used for the analysis was set at 0.5%, it was theoretically detectable from 1% sample, but in practice, 5% sample could be detected. These studies have shown that "what is the sensitivity (sensitivity) and how precise (specificity) is the detection of the allele from the minor contributor", which is of greatest interest in analyzing mixtures using NGS I think it will provide important information to learn. In the future, it will be necessary to try to make accurate interpretation of the data obtained by analyzing NGS data to determine STR alleles.

As a strategy of NGS data analysis proposed in this study, a new method is proposed to determine the allele genotype according to the STR locus used mainly in forensic science. A lobSTR program has been reported to analyze STR previously developed by other researchers. However, the method presented in this study showed better results than the lobSTR program (data not shown). The analytical methods used in this study seem to be somewhat difficult for actual users to feel because of the complicated and cumbersome process. Therefore, if an analysis program using the newly generated reference sequence is developed, NGS analysis will be performed more efficiently and it will be useful for application of more STR analysis.

In this study, it was possible to successfully generate sequencing data for single male and female standard samples and their mixed samples through one NGS process, and to perform effective STR analysis from these data. This method is considered to be applicable to the analysis of samples collected from suspects and victims together with the samples found at the crime scene. Therefore, if the STR analysis using NGS is more optimized in terms of experimental and analytical aspects, it can be used as an additional method in addition to existing methods in the forensic science field by satisfying the deficiencies of the existing CE-based method.

la. references

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Example  2: Multiplex Gene Amplification for the Analysis of Human Objects in Mixed Samples Autosomal  analysis

end. Research method

1. Single-source ( single - source ) And mixing DNA  Sample

DNA samples were obtained from a male standard sample 2800M (Promega, Madison, WI, USA) and a female standard sample 9947A (Promega), which are used as control in legal genetics research. These DNA samples were quantified using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) and then prepared at a concentration of 1 ng / μl. Mixed samples were prepared by mixing two single samples (2800M and 9947A) at a ratio of 1: 1, 1: 3, 1: 6, 1: 9 and 1:49 (male: female), respectively, to a final concentration of 1 ng / μl Ready.

2. Amplification method

In this study, a novel multiplex PCR system constructed for STR NGS analysis was used to amplify single source samples and mixtures. The system includes 18 markers (CODIS 13 STRs, D2S1338, D19S433, Penta D, Penta E, and ammelogenin) generally used in forensic genetics. Primers were designed using the Primer 3 program for amplification of the STR region. The amplicon size ranged from 70 bp to 210 bp, and no fluorescent dye was used. We designed the primers so that no more than 1% mutation appears at the primer binding site based on the NCBI SNP information ( http://www.ncbi.nlm.nih.gov/SNP/ ).

PCR primers and final concentrations of 18 markers in-house developed multiplex PCR system - Locus Primer set The primer sequence (5` → 3`) Concentration (μM) One D19S433 F053 GCAAAAAGCTATAATTGTACCAC 0.60 R203 AAAAATCTTCTCTCTTTCTTCCTCTC 0.60 2 D5S818 F160 TGATTTTCCTCTTTGGTATCCTT 0.55 R280 CAACATTTGTATCTTTATCTGTATCCT 0.55 3 Penta E F203 GGCGACTGAGCAAGACTCA 1.00 R284m TGGGTTATTAATTGAGAAAACTCCTT 1.00 4 CSF1PO F191 ACTGCCTTCATAGATAGAAGAT 0.50 R295 GACCCTGTTCTAAGTACTTCCT 0.50 5 D7S820 F171 TGATAGAACACTTGTCATAGTTTAGAA 0.50 R344 CTCATTGACAGAATTGCACCA 0.50 6 D18S51 F197 GTTGCTACTATTTCTTTTCTTTTTCTC 0.90 R340 CTGAGTGACAAATTGAGACCTTG 0.90 7 TPOX F112 CAGAACAGGCACTTAGGGAAC 0.35 R198 TCCTTGTCAGCGTTTATTTGC 0.35 8 D16S539 F119 AATACAGACAGACAGACAGGTG 0.45 R225 AGCATGTATCTATCATCCATCTCTG 0.45 9 D8S1179 F173 TTTTTGTATTTCATGTGTACATTCGT 0.90 R275 GTAGATTATTTTCACTGTGGGGAA 0.90 10 Amelogenin F181 CCTTTGAAGTGGTACCAGAGCAT 0.80 R262 GCATGCCTAATATTTTCAGGGAATAA 0.80 11 FGA F153 AAATAAAATTAGGCATATTTACAAGC 1.00 R293 GCCAGCAAAAAAGAAAGGAA 1.00 12 D13S317 F183 TCTAACGCCTATCTGTATTTACAA 0.80 R284 AGACAGAAAGATAGATAGATGATTGA 0.80 13 D2S1338 F128 TGGAAACAGAAATGGCTTGG 0.80 R273 AGTTATTCAGTAAGTTAAAGGATTGC 0.80 14 D21S11 F161 AATTCCCCAAGTGAATTGCC 0.70 F334 GGTAGATAGACTGGATAGATAGACGA 0.70 15 Penta D F153 GCAAGACACCATCTCAAGAAAG 1.00 R318 TGGTCATAACGATTTTTTTGAGA 1.00 16 D3S1358 F145 CAGTCCAATCTGGGTGACAG 0.50 R266 ATCAACAGAGGCTTGCATGT 0.50 17 TH01 F117 GATTCCCATTGGCCTGTTC 0.50 R216 CAGGTCACAGGGAACACAGA 0.50 18 vWA F096 GAATAATCAGTATGTGACTTGGATTG 1.00 R226 TGATAAATACATAGGATGGATGG 1.00

Multiplex PCR reactions included 1 ng template DNA, 4.5 U AmpliTaq Gold DNA polymerase (Applied Biosystems), 2.5 μL Gold ST * R 10 × buffer (Promega) and 0.35 μM-1.0 μM primers (Table 7) 25 [mu] L reaction volume. The PCR cycle was performed using Veriti 96-Well Thermal Cycler (Applied Biosystems) under the following conditions: 95 ° C, 11 min; 33 cycles, 94 캜 (20 sec), 59 캜 (90 sec) and 72 캜 (60 sec); And final extension, 60 < 0 > C (45 min); 4 ° C.

After PCR, 25 μL of the PCR product was purified by treating with 10 μL of ExoSAP IT (USB, Cleveland, OH, USA) for 45 minutes at 37 ° C., followed by heat treatment at 80 ° C. for 15 minutes to inactivate the PCR product. The purified enzyme-purified PCR product was further purified using Qiaquick PCR Purification Kit (Qiagen, Hilden, Germany). Finally, the size distribution was analyzed using a Bioanalyzer 2100 using an Agilent High Sensitivity DNA Kit (Agilent Technologies, Palo Alto, Calif., USA) and the purity using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, Mass., USA) , And the quality of the purified ampicillin was calculated by quantification using Quant-iT ™ PicoGreened DNA Assay Kit (Invitrogen, Carlsbad, Calif., USA).

3. Artificially decomposed DNA of PCR  Amplification

An artificially degraded sample was prepared by adding 0.006 U of DNase I (New England Biolabs, Ipswich, MA, USA) to K562 polymer DNA (Promega) and reacting at 37 ° C for 15 minutes and 75 ° C for 10 minutes.

DNA fragments were confirmed by agarose gel electrophoresis. The amplification reaction was carried out under the same conditions as described above under a reaction volume of 25 μL containing 200 pg of the digested DNA and 6.0 U of AmpliTaq Gold DNA polymerase (Applied Biosystems), and the PCR reaction was carried out at 36 cycles. All experiments were conducted in duplicate and labeled AD01 and AD02, respectively.

4. Prepare your library and NGS  Data generation

From 200 ng or more purified PCR products, libraries were generated by the TruSeqNano DNA Sample Preparation Kit (Illumina). All procedures were performed according to the manufacturer's method, and the beads ratio for sizing was controlled in the bead refining process based on previous work [19].

The completed library was quantified using the qPCR method presented in Illumina. The Bioanalyzer was used to confirm the size distribution and to measure the total concentration of the final library. Samples were sequenced on MiSeq ™ (Illumina Inc., San Diego, Calif., USA) and automatically sorted by the MiSeq Reporter.

5. MiSeq  Data Analysis

STR alleles were retrieved from MiSeq data according to the protocol described in Bornman et al. [6]. Briefly, we constructed an STR reference sequence in the FASTA format by linking it to a recently known allele sequence and a 5 'and 3' flanking sequence. The allele sequence was obtained from STRbase ( http://www.cstl.nist.gov/strbase ). Flanking sequences of 500 bp to 550 bp in length were obtained from the human reference genome GRCh37 / hg19. Four programs (Bowtie 2 [20], SAMtools [21], BEDTools [22], and Microsoft Excel) were used for STR profiling from NGS data. I) the STR locus name of each allele, ii) the allele name, and iii) the start of the repeat site, in order to detect sequence leads spanning 5 bp of the entire STR region and 5 'and 3' flanking sequences. And a tab-delimited BED file containing the end point.

Sequence reads of MiSeq data were aligned on the STR reference sequence using the Bowtie 2 program. We then used SAMtools and BEDTools to convert the format of the alignment results file. Finally, using Microsoft Excel, STR alleles were determined in each locus by counting the number of sequence leads containing the entire STR region from the alignment results and the BED file. During this process, 20% of the total coverage value was used in the STR allele call of the single-source sample, and 10%, 5%, and 1% were applied to the mixture samples at various mixing ratios. Threshold values were determined empirically by analyzing the coverage of STR alleles in each locus for the entire data set. In order to measure STR allele call results, MiSeq data were analyzed using the STRait Razor program [17]. Sequence variations in the target STR region were analyzed by comparing the reference and sample sequences using the Integrative Genomics Viewer [23,24], and then the NCBI dbSNP ( http://www.ncbi.nlm.nih.gov) / projects / SNP / ).

6. For comparison CE  Profile data

All samples were detected as AmpFlSTR IdentifilerKit (Applied Biosystems) and analyzed with ABI GeneMapperID Software Version 3.2 via ABI 3130xl Genetic Analyzer with Data Collection Software Version 3.0. The STR profile obtained by this method was used as reference data for comparison with STR type results from NGS data. For artificially segmented DNA, 200 pg of template DNA was used and thermal cycling was corrected to 33 cycles.

I. Results

1. Construction of an in-house multiplex PCR system optimized for NGS analysis

The in-house multiplex PCR system consists of 13 CODIS STR loci, 18 loci for NGS analysis, including 4 loci (D2S1338, D19S433, Penta D and Penta E) and ammelogenin for use in general forensic commercial kits Was built. Amplicons are designed in sizes from 70 bp to 210 bp (Fig. 4) and are compatible with the lead lengths of currently available NGS platforms. To determine the degree of primer interference, PCR amplicons were analyzed with dye-labeled primers. As a result, the final primer set on the electrophorogram showed balanced amplification efficiency without any primer interference (FIG. 6).

2. Sequencing data

Table 8 shows the number of aligned lead numbers across all repeat sites of a particular STR allele in each sample. 30 (Q30) has a quality score of 75.8%. The coverage of each allele was classified according to 18 markers and aligned with the reference sequence (Fig. 5). The total coverage according to STR loci showed a minimum of 29,818 leads in CSF1PO and a maximum of 107,477 leads in D2S1338. In conclusion, it is possible to group the alleles according to the number of repeats, which allows the sequence ratio of the stutter, which is a small number of PCR products, which is as short as one iteration than the allele and the parent allele to be calculated.

The number of aligned leads of each sample from MiSeq data Sample 2800M 9947A 1: 1 mixture 1: 3 mixture Paired-end read Lead 1 Lead 2 Lead 1 Lead 2 Lead 1 Lead 2 Lead 1 Lead 2 All leads 1,046,963 1,037,184 1,109,040 1,100,166 1,001,233 990,550 1,008,585 1,000,252 Sample 1: 6 mixture 1: 9 mixture 1:19 mixture 1:49 mixture Paired-end read Lead 1 Lead 2 Lead 1 Lead 2 Lead 1 Lead 2 Lead 1 Lead 2 All leads 1,045,519 1,037,230 1,006,843 997,143 1,795,434 1,776,801 2,302,059 2,285,333

3. Single-source and mixed samples STR Zino typing

Using the obtained NGS data, most STR alleles and amelogenins can be successfully determined by applying a 20% coverage threshold of a single-source sample. Next, the STR genotype of the 18 markers was the same for CE and NGS based analyzes (Table 9).

Two Single Source STR Genotype Typing Results from NGS Data STR locus 2800M 9947A CE NGS CE NGS D2S1338 22, 25 22, 25 19, 23 19, 23 D3S1358 17, 18 17, 18 14, 15 14, 15 D5S818 12 12 11 11 D7S820 8, 11 8, 11 10, 11 10, 11 D8S1179 14, 15 14, 15 13 13 D13S317 9, 11 9, 11 11 11 D16S539 9, 13 9, 13 11, 12 11, 12 D18S51 16, 18 16, 18 15, 19 15, 19 D19S433 13, 14 13, 14 14, 15 14, 15 D21S11 29, 31.2 29, 31.2 30 30 CSF1PO 12 12 10, 12 10, 12 FGA 20, 23 20, 23 23, 24 23, 24 Penta D 12, 13 12, 13 12 12 Penta E 7, 14 7, 14 12, 13 12, 13 TH01 6, 9.3 6, 9.3 8, 9.3 8, 9.3 TPOX 11 11 8 8 vWA 16, 19 16, 19 17, 18 17, 18 Amelogenin X, Y X, Y X X

The results of STR genotyping of the mixed samples by NGS are shown in Table 10. This result almost coincides with the CE-based profile. Interestingly, alleles lower than the coverage threshold (10%) observed as a drop-out in a 1: 1 mixture were 9947A genotype. However, the results for the 1: 6 and 1:49 mixtures showed an allele of 2800M genotype lower than the coverage threshold (#% and #%, respectively). A drop-in allele was observed in the data, some of which were more than 15% of the stutter threshold.

Mixture STR NG Typing Results from NGS Data STR locus MiSeq STR data 1: 1 1: 3 1: 6 1: 9 1:19 1:49 D2S1338 19, 22, (23) ^a , 25 19, 22, 23, 25 19, 22, 23, 25 18 ^b , 19, 22, 23, 25 19, 22, 23, 25 18 ^b , 19, 22, 23, (25) ^a D3S1358 14, 15, 17, 18 14, 15, 17, 18 14, 15, 17, 18 14, 15, 17, 18 14, 15, 17, 18 14, 15, 17, (18) ^a D5S818 11, 12 11, 12 11, 12 11, 12 11, 12 11, 12 D7S820 8, 10, 11 8, 10, 11 8, 10, 11 8, 10, 11 8, 10, 11 8, 10, 11 D8S1179 13, 14, 15 13, 14, 15 13, 14, 15 13, 14, 15 13, 14, 15 11 ^b , 13, 14, 15 D13S317 9, 11 9, 11 9, 11 9, 11 9, 11 9, 11 D16S539 9, (11) ^a , 12, 13 9, 11, 12, 13 9, 11, 12, 13 9, 11, 12, 13 9, 11, 12, 13 9, 11, 12, 13 D18S51 15, 16, 18, 19 15, 16, 18, 19 15, 16, 18, 19 14 ^b , 15, 16, 18, 19 14 ^b , 15, 16, 18, 19 12 ^b , 13 ^b , 14 ^b , 15, (16) ^a , 18, 19 D19S433 13, 14, (15) ^a 13, 14, 15 13, 14, 15 13, 14, 15 13, 14, 15 13, 14, 15 D21S11 29, 30, 31.2 29, 30, 31.2 29, 30, 31.2 29, 30, 31.2 29, 30, 30.2 ^b , 31.2 29, 29.2 ^b , 30, 30.2 ^b , 31.2 CSF1PO (10) ^a , 12 10, 12 10, 12 10, 12 10, 12 10, 12 FGA 20, 23, 24 20, 23, 24 (20) ^a , 23, 24 20, 23, 24 20, 23, 24 (20) ^a , 23, 24 Penta D 12, 13 12, 13 12, 13 12, 13 12, 13 12, 13 Penta E 7, (12) ^a , 13, 14 7, 12, 13, 14 7, 12, 13, (14) ^a 7, 12, 13, 14 7, 12, 13, 14 (7) ^a , 12, 13, 14 TH01 6, (8) ^a, 9.3 6, 8, 9.3 6, 8, 9.3 6, 8, 9.3 6, 8, 9.3 6, 8, 9.3 TPOX 8, 11 8, 11 8, 11 8, 11 8, 11 8, 11 vWA 16, 17, 18, 19 16, 17, 18, 19 16, 17, 18, 19 16, 17, 18, 19 16, 17, 18, 19 16, 17, 18, (19) ^a , 25 ^b coverage threshold
10% 10% 10% 5% 2.5% One%

a Alleles in parentheses indicate alleles with coverers below each coverage threshold.

b The underlined allele represents an unexpected allele with a value higher than the stutter threshold.

4. Observed sequence variations

In the sequencing data, sequence variations from repeating sites of D2S1338, D8S1179, D21S11, D3S1358, and vWA were observed. The D2S1338 allele sequence is shown as [TGCC] a [TTCC] b. Sequence variation of D2S1338 at 9947A was observed in the interconnecting unit 'GTCC' (Fig. 7A). This mutation type has been reported in NCBI as a single nucleotide polymorphism (SNP), rs9678338 (T> G). The allele sequence of D3S1358 is designated TCTA [TCTG] a [TCTA] b. Sequence variation was observed in the second sub-repeat unit 'TCTA'. These mutations were reported as rs77577482 (A> G) and rs71325067 (A> G), respectively. The allele sequence of D8S1179 is shown as [TCTA] a [TCTG] b [TCTA] c. Sequence variation of D8S1179 was observed in the interconnection unit 'TCTG' and the sub-repeat unit 'TCTA'. Interestingly, the 9947A allele was identified as a heterozygous allele by NGS data whereas the CE was a homozygous allele (Fig. 7c). As a result, the present inventors could identify the same allele according to the sequence variation by NGS. These mutations were reported as rs13265375 (G> A) and rs111782616 (A> G), respectively. In the D21S11 locus, three sequence mutations previously reported in Danes [11,13] were identified. Compared to the NCBI SNP database, the mutations were identified as rs13049099 (G> A), rs200026324 (G> A) and rs13050496 (A> G) (Fig. 7d). The allele sequence of vWA may be represented as TCTA [TCTG] a [TCTA] b TCCA TCTA. Sequence variation of vWA was observed in the interconnected unit 'TCTG'.

6. Artificially segmented DNA

The present inventors analyzed artificially segmented DNA samples. However, non-specific peaks were observed in the electrophorogram of the Bioanalyzer phase library, and the effect on NGS analysis was not revealed (data not shown). As a result, the sample profile was generally consistent between duplicate amplifications, consistent with the partial CE profile amplification obtained with the Identifiler kit (Table 11). However, some alleles showed an allele drop-out in the CE profile (D13S317, D18S51: AD01; D2S1338, D7S820: AD02). On the other hand, the allele drop-in was found in some STR sites when the 10% coverage threshold was applied to determine the STR allele.

STR Genotyping results of artificially degraded DNA from NGS data STRs K562 profile AD01 AD02 CE NGS CE NGS D2S1338 17 17 17 - 17 D3S1358 16 16 15 ^b , 16 16 16 D5S818 11, 12 11, 12 11, 12 11 11, 12 D7S820 9, 11 9, 11 9, 11 - 9, 10 ^b , 11 D8S1179 12 12 12 12 11 ^b , 12 D13S317 8 - 8 8 8 D16S539 11, 12 11, 12 11, 12 12 11, 12 D18S51 15, 16 - 14 ^b , 15, 16 15, 16 14, 15, 16 D19S433 14, 14.2 14, 14.2 14, 14.2 14.2 14, 14.2 D21S11 29, 30, 31 31 29, (30) _a , 31 31 29, 30, 31 CSF1PO 9, 10 9 9, 10 10 9, 10 FGA 21, 24 21, 24 21, 24 21, 24 21, 24 Penta D 9, 13 - 9, 13 - 9, 13 Penta E 5, 14 - 5, 14 - 5, 14 TH01 9.3 9.3 9.3 9.3 9.3 TPOX 8, 9 8, 9 8, 9 8, 9 8, 9 vWA 16 16 16 16 16, 18 ^b Amelogenin X X X X X

a Alleles in parentheses represent alleles with a coverager value lower than the 10% coverage threshold.

b The underlined allele represents an unexpected allele with a value higher than the stutter threshold.

Based on the leader for the allele, we have identified an allele thought to be a stutter of the true allele, which appeared as an allele drop-in. This is because their coverage is somewhat higher than the stutter analysis threshold. In conclusion, the overlapping results of NGS showed more STR profiles than the CE profiles with high coverage.

All. Review

Using an in-house multiplex PCR system, we have demonstrated that it is possible to generate NGS data sets, determine STR alleles, and detect sequence variations in the STR region. The system was designed to analyze forensic STR loci using NGS and can generate small amplicons within a narrow size range of 70 bp to 210 bp using non-labeled primers. Because the MiSeq platform can generate sequencing leads with an average length of 200 bp, this system can generate STR NGS data of excellent quality. The system can also be applied to forensic samples. The STR presented in our multiplex PCR system is significant if we can obtain a consistent PCR product that balances STR loci with no more than 34 primers and no primer interference

In this study, CE profiling in a mixture of single-source samples and two control DNA samples (* 1: 1, 1: 3, 1: 6, 1: 9, 1:19, and 1:49) . When the CE profile of the mixture was observed, the electrophoresis did not distinguish the peak and noise of the PCR amplification product. NGS data confirmed that true alleles could be identified based on the number of read numbers. Despite the presence of PCR efficiency in the sample, we observed accurate genotyping results in nearly half of STR loci: D5S818, D7S820, D13S317, D16S539, D19S433, CSF1PO, Penta D, TH01, TPOX - 1:49 mixture . However, the mixture analysis was tested with only two donors using control DNA, not casework samples. The present inventors also failed to provide a lower limit of 1:49 mixing ratio in NGS.

We have identified sequence variations at the STR site (D2S1338, D3S1358, D8S1179, D21S11 and vWA). These variations were reported in the SNP database. These mutations allow to distinguish individuals with alleles of the same length. This means that the discrimination power (PD) is increased. Mixture analysis using substantially two control DNAs was distinguishable by observing the same allele, not other sequence variants. Whereby alleles within the same mixture can be distinguished by observed sequence variations.

In order to compare with the obtained sequence variation data, the present inventors retrieved previously disclosed data. Up to now no nomenclature has been established for sequence variation in STR regions. One study has pointed to this problem [13]. A new nomenclature of the sequenced STR profile is required for STR data consistency from NGS.

We also analyzed artificially degraded DNA as forensic case work samples. Compared to the CE profile of artificially segmented DNA, NGS data gave more useful information. As the amplification strategy for small size products was established, the STR profile was successfully obtained. However, the present inventors could not estimate the state of true case work samples. In addition, analyzing case work samples such as skeletal residues and soil exposed samples, this method will provide more useful information from forensically medically challenging case work samples and may prove their usefulness.

The Forensic Academy wanted to analyze the base composition by sequencing using mass spectrometry. Studies using forensic STR have already been done [25-28]. Sequence variations were also observed at the repeat sites. However, their location in the sequence was not known, and Snager sequencing was performed as an additional analysis step. For this reason, NGS has received much attention in the forensic field. The use of NGS in forensic science has provided additional tools for CE-based methods and has been studied more intensively. Mitochondrial DNA, Y STRs, and X-STRs as well as forensic STRs can be analyzed on the NGS platform. Therefore, this study will provide a reference study that can be optimized in multiplex PCR by considering NGS lead length. We sequenced two control DNAs as single-source samples. In previous studies that analyzed STR using mass spectrometry, the amount of information increased was to reveal additional allelic variants. Observed mutations mean increased discrimination and efficacy.

Many researchers say NGS experiments are simpler than ever before. However, many steps are required to analyze the STF from the amplification reaction using 1 ng of sample to the sequencing of NGS. The inventors have found that creating a library when using the TruSeq system on the MiSeq platform is a more difficult and time consuming task than the CE method. In particular, the number of libraries generated at the same time is small. If you want to use NGS as an additional powerful tool in public forensic laboratories, it is necessary to simplify the experimental steps.

Although the present inventors used only the MiSeq platform, the in-house developed multiplex PCR system provides superior quality when applied to another NGS platform and provides another suitable evelo. Thus, in-house developed multiplex PCR systems are appropriate for specific NGS platforms. Use this to make additional experiments, including sensitivity testing, on various NGS platforms. If you perform additional experiments and supplement STR NGS studies, this can be useful for forensic genetics.

In addition, the NGS analysis of STR using the multiplex PCR system constructed in this study is expected to be useful not only for single samples but also for analyzing mixed samples. It is expected that the analysis of the mixed sample can be performed more easily.

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[29] JM Butler, Advanced topics in forensic DNA typing: methodology, Elsevier academic press, New York, 2011.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Republic Of Korea (Supreme Public Prosecutor's Office) <120> Method for Autosomal Analysis Human Subject of Analytes based on          a Next Generation Sequencing Technology <130> PN140529 <160> 68 <170> Kopatentin 2.0 <210> 1 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> D3S1358 forward primer <400> 1 actgcagtcc aatctgggt 19 <210> 2 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> D3S1358 reverse primer <400> 2 atgaaatcaa cagaggcttg c 21 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TH01 forward primer <400> 3 gtgattccca ttggcctgtt c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> TH01 reverse primer <400> 4 attcctgtgg gctgaaaagc tc 22 <210> 5 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> D21S11 forward primer <400> 5 atatgtgagt caattcccca ag 22 <210> 6 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D21S11 reverse primer <400> 6 tgtattagtc aatgttctcc agagac 26 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D18S51 forward primer <400> 7 ttcttgagcc cagaaggtta 20 <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> D18S51 reverse primer <400> 8 attctaccag caacaacaca aataaac 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Penta E forward primer <400> 9 attaccaaca tgaaagggta ccaata 26 <210> 10 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Penta E reverse primer <400> 10 tgggttatta attgagaaaa ctccttacaa ttt 33 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> D5S818 forward primer <400> 11 ggtgattttc ctctttggta tcc 23 <210> 12 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D5S818 reverse primer <400> 12 agccacagtt tacaacattt gtatct 26 <210> 13 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D13S317 forward primer <400> 13 attacagaag tctgggatgt ggagga 26 <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> D13S317 reverse primer <400> 14 ggcagcccaa aaagacaga 19 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> D7S820 forward primer <400> 15 atgttggtca ggctgactat g 21 <210> 16 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D7S820 reverse primer <400> 16 gattccacat ttatcctcat tgac 24 <210> 17 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D16S539 forward primer <400> 17 gggggtctaa gagcttgtaa aaag 24 <210> 18 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> D16S539 reverse primer <400> 18 gtttgtgtgt gcatctgtaa gcatgtatc 29 <210> 19 <211> 24 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > CSF1PO forward primer <400> 19 ccggaggtaa aggtgtctta aagt 24 <210> 20 <211> 22 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > CSF1PO reverse primer <400> 20 atttcctgtg tcagaccctg tt 22 <210> 21 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Penta D forward primer <400> 21 gaaggtcgaa gctgaagtg 19 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> Penta D reverse primer <400> 22 attagaattc tttaatctgg acacaag 27 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> vWA forward primer <400> 23 gccctagtgg atgataagaa taatcagtat gtg 33 <210> 24 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> vWA reverse primer <400> 24 ggacagatga taaatacata ggatggatgg 30 <210> 25 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> D8S1179 forward primer <400> 25 attgcaactt atatgtattt ttgtatttca tg 32 <210> 26 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> D8S1179 reverse primer <400> 26 accaaattgt gttcatgagt atagtttc 28 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TPOX forward primer <400> 27 gcacagaaca ggcacttagg 20 <210> 28 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> TPOX reverse primer <400> 28 cgctcaaacg tgaggttg 18 <210> 29 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FGA forward primer <400> 29 ggctgcaggg cataacatta 20 <210> 30 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> FGA reverse primer <400> 30 attctatgac tttgcgcttc agga 24 <210> 31 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin forward primer <400> 31 ccctgggctc tgtaaagaa 19 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin reverse primer <400> 32 atcagagctt aaactgggaa gctg 24 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> D19S433 forward primer <400> 33 gcaaaaagct ataattgtac cac 23 <210> 34 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D19S433 reverse primer <400> 34 aaaaatcttc tctctttctt cctctc 26 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> D5S818 forward primer <400> 35 tgattttcct ctttggtatc ctt 23 <210> 36 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> D5S818 reverse primer <400> 36 caacatttgt atctttatct gtatcct 27 <210> 37 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Penta E forward primer <400> 37 ggcgactgag caagactca 19 <210> 38 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Penta E reverse primer <400> 38 tgggttatta attgagaaaa ctcctt 26 <210> 39 <211> 22 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > CSF1PO forward primer <400> 39 actgccttca tagatagaag at 22 <210> 40 <211> 22 <212> DNA <213> Artificial Sequence <220> &Lt; 223 > CSF1PO reverse primer <400> 40 gaccctgttc taagtacttc ct 22 <210> 41 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> D7S820 forward primer <400> 41 tgatagaaca cttgtcatag tttagaa 27 <210> 42 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> D7S820 reverse primer <400> 42 ctcattgaca gaattgcacc a 21 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> D18S51 forward primer <400> 43 gttgctacta tttcttttct ttttctc 27 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> D18S51 reverse primer <400> 44 ctgagtgaca aattgagacc ttg 23 <210> 45 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TPOX forward primer <400> 45 cagaacaggc acttagggaa c 21 <210> 46 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> TPOX reverse primer <400> 46 tccttgtcag cgtttatttg c 21 <210> 47 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> D16S539 forward primer <400> 47 aatacagaca gacagacakg tg 22 <210> 48 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> D16S539 reverse primer <400> 48 agcatgtatc tatcatccat ctctg 25 <210> 49 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D8S1179 forward primer <400> 49 tttttgtatt tcatgtgtac attcgt 26 <210> 50 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D8S1179 reverse primer <400> 50 gtagattatt ttcactgtgg ggaa 24 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin forward primer <400> 51 cctttgaagt ggtaccagag cat 23 <210> 52 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> Amelogenin reverse primer <400> 52 gcatgcctaa tattttcagg gaataa 26 <210> 53 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> FGA forward primer <400> 53 aaataaaatt aggcatattt acaagc 26 <210> 54 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> FGA reverse primer <400> 54 gccagcaaaa aagaaaggaa 20 <210> 55 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> D13S317 forward primer <400> 55 tctaacgcct atctgtattt acaa 24 <210> 56 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D13S317 reverse primer <400> 56 agacagaaag atagatagat gattga 26 <210> 57 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D2S1338 forward primer <400> 57 tggaaacaga aatggcttgg 20 <210> 58 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D2S1338 reverse primer <400> 58 agttattcag taagttaaag gattgc 26 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D21S11 forward primer <400> 59 aattccccaa gtgaattgcc 20 <210> 60 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> D21S11 reverse primer <400> 60 ggtagataga ctggatagat agacga 26 <210> 61 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Penta D forward primer <400> 61 gcaagacacc atctcaagaa ag 22 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Penta D reverse primer <400> 62 tggtcataac gatttttttg aga 23 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D3S1358 forward primer <400> 63 cagtccaatc tgggtgacag 20 <210> 64 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> D3S1358 reverse primer <400> 64 atcaacagag gcttgcatgt 20 <210> 65 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> TH01 forward primer <400> 65 gattcccatt ggcctgttc 19 <210> 66 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> TH01 reverse primer <400> 66 caggtcacag ggaacacaga 20 <210> 67 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> vWA forward primer <400> 67 gaataatcag tatgtgactt ggattg 26 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> vWA reverse primer <400> 68 tgataaatac ataggatgga tgg 23

Claims (33)

An autosomal analysis method of an NGS-based next generation sequencing-based human subject comprising the steps of:
(a) Human DnaS1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, vWA Multiplex amplification using respective primers complementarily binding to the genetic locus of D8S1179, human thyroid peroxidase gene (TPOX), human fibrinogen alpha chain (FGA) and amelogenin, Wherein the primer complementarily binding to the D3S1358 genetic locus is a first sequence and a second sequence of Sequence Listing; The primer that binds complementarily to the TH01 genetic locus is a sequence of SEQ ID NO: 3 or SEQ ID NO: 4; The primers complementarily binding to the D21S11 genetic locus are those of SEQ ID NOS: 5 and 6; The primers complementarily binding to the D18S51 genetic locus are SEQ ID NOS: 7 and 8; Wherein the primer that binds complementarily to the PentaE gene locus is SEQ ID NOS: 9 and 10; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 11 and SEQ ID NO: 12; Wherein the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS 13 and 14; The primers that complementarily bind to the D7S820 genetic locus are those of Sequence Listing 15 and 16; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 17 and Sequence 18; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NOS: 19 and 20; Wherein the primers complementarily binding to the PentaD genetic locus are SEQ ID NOS 21 and 22, and the primers complementarily binding to the vWA genetic locus are SEQ ID NOS 23 and 24; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 25 and Sequence 26; Wherein the primer that binds complementarily to the TPOX genetic locus is SEQ ID NO: 27 and SEQ ID NO: 28; Wherein the primers complementarily binding to the FGA genetic locus are those of Sequence Listing Nos. 29 and 30; Wherein the primers complementarily binding to the amelogenin gene locus are SEQ ID NOS: 31 and 32; And
(b) determining an STR (short tandem repeat) allele of the genetic locus using the NGS data of the multiplex amplification product of step (a), and identifying the human object as a gene.
The method of claim 1, wherein the DNA sample is DNA isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or amniotic fluid including fetal cells, Wherein the sample is a sample.
The method according to claim 1, wherein the DNA sample is a biological sample containing DNA.
The method according to claim 1, wherein the multiplex gene amplification is a Polymerase Chain Reaction (PCR) amplification or a direct multiplex PCR amplification.
5. The method according to claim 4, wherein the multiplex gene Wherein the amplification has 32-36 cycles.
delete 2. The method according to claim 1, wherein the primer complementarily binding to the D3S1358 genetic locus of step (a) has a final concentration of 0.01-0.5 [mu] M and the primer that binds complementarily to the TH01 genetic locus is 0.01-0.4 [mu] M Wherein the primer complementarily binding to the D21S11 genetic locus has a final concentration of 0.4-0.8 μM and the primer complementarily binding to the D18S51 genetic locus has a final concentration of 0.3-0.7 μM, The primer complementarily binding to the PentaE gene locus has a final concentration of 1-1.4 [mu] M, and the primer complementarily binding to the D5S818 genetic locus has a final concentration of 0.01-0.5 [mu] M, and the D13S317 gene locus is complementary The primer that binds as a target has a final concentration of 0.2-0.6 [mu] M; The primer that binds complementarily to the D7S820 genetic locus has a final concentration of 0.1-0.5 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.2-0.6 [mu] M; The primer that binds complementarily to the CSF1PO genetic locus has a final concentration of 0.1-0.5 [mu] M; The primer that binds complementarily to the PentaD genetic locus has a final concentration of 1-1.4 [mu] M; The primer that binds complementarily to the vWA genetic locus has a final concentration of 0.01-0.4 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.3-0.7 [mu] M; The primer that binds complementarily to the TPOX genetic locus has a final concentration of 0.01-0.4 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.4-0.8 [mu] M; Wherein the primer complementarily binding to the amelogenin gene locus has a final concentration of 0.05-0.5 [mu] M.
2. The method according to claim 1, wherein step (b) comprises comparing the nucleotide sequence of each STR allele amplified in the multiplex amplification product with a reference sequence, Lt; RTI ID = 0.0 &gt; STR &lt; / RTI &gt;
[Claim 9] The method according to claim 8, wherein the reference sequence comprises a sequence of the STR region of the allele, a 5 'perimeter sequence of the STR region, and a 3' peripheral sequence of the STR region.
9. The method of claim 8, wherein the method further comprises, after step (b), identifying the number of copies of the repeat sequence, the repeat structure of alleles, or sequence variation in the STR genomic locus RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
11. The method of claim 10, wherein the method further comprises the step of measuring the mixing ratio of the mixed sample using the sequence variation, wherein the mixing ratio is determined by analyzing coverage ratio between alleles on each STR gene, Lt; RTI ID = 0.0 &gt; mutation &lt; / RTI &gt; base sequence.
2. The method of claim 1, wherein the multiplex amplification product of step (b) has a size of 20 bp to 600 bp.
2. The method of claim 1, wherein the method of autosomal analysis has the use of forensic typing or identification.
2. The method of claim 1, wherein the primer is a non-labeled primer.
D3S1358, TH01, D21S11, D18S51, PentaE, D5S818, D13S317, D7S820, D16S539, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), PentaD, von Willebrand factor A, D8S1179, TPOX (next generation sequencing-based) autosomal multiplex multiplex gene amplification that includes primers complementary to the thyroid peroxidase gene, FGF (human fibrinogen alpha chain), and amelogenin gene locus As a kit, the primer that binds complementarily to the D3S1358 genetic locus is a first sequence and a second sequence of Sequence Listing; The primer that binds complementarily to the TH01 genetic locus is a sequence of SEQ ID NO: 3 or SEQ ID NO: 4; The primers complementarily binding to the D21S11 genetic locus are those of SEQ ID NOS: 5 and 6; The primers complementarily binding to the D18S51 genetic locus are SEQ ID NOS: 7 and 8; Wherein the primer that binds complementarily to the PentaE gene locus is SEQ ID NOS: 9 and 10; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 11 and SEQ ID NO: 12; Wherein the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS 13 and 14; The primers that complementarily bind to the D7S820 genetic locus are those of Sequence Listing 15 and 16; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 17 and Sequence 18; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NOS: 19 and 20; Wherein the primers complementarily binding to the PentaD genetic locus are SEQ ID NOS 21 and 22, and the primers complementarily binding to the vWA genetic locus are SEQ ID NOS 23 and 24; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 25 and Sequence 26; Wherein the primer that binds complementarily to the TPOX genetic locus is SEQ ID NO: 27 and SEQ ID NO: 28; Wherein the primers complementarily binding to the FGA genetic locus are those of Sequence Listing Nos. 29 and 30; Wherein the primers complementarily binding to the amelogenin gene locus are SEQ ID NOS: 31 and 32.
An autosomal analysis method of an NGS-based next generation sequencing-based human subject comprising the steps of:
(a) Human DnaSDNA, D5S818, Penta E, CSF1PO (Human c-fms proto-oncogene for CSF-1 receptor gene), D7S820, D18S51, TPOX (human thyroid peroxidase gene), D16S539, D8S1179, Amplifier amplification was performed using primers complementary to the genetic locus of amelogenin, human fibrinogen alpha chain (FGA), D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA (von Willebrand factor A) Wherein the primer that binds complementarily to the D19S433 genetic locus is SEQ ID NO: 33 and SEQ ID NO: 34; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 35 and SEQ ID NO: 36; Wherein the primer that binds complementarily to the Penta E gene locus is a sequence of SEQ ID NOS: 37 and 38; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NO: 39 and SEQ ID NO: 40; The primers complementarily binding to the D7S820 genetic locus are those of Sequence Listing 41 and Sequence 42; The primers complementarily binding to the D18S51 genetic locus are those of Sequence Listing 43 and Sequence 44; Wherein the primers complementarily binding to the TPOX genetic locus are those of Sequence Listing 45 and 46; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 47 and Sequence 48; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 49 and Sequence 50; Wherein the primers complementarily binding to the amelogenin gene locus are SEQ ID NO: 51 and SEQ ID NO: 52; Wherein the primers complementarily binding to the FGA gene locus are SEQ ID NOS: 53 and 54, and the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS: 55 and 56; The primers complementarily binding to the D2S1338 genetic locus are those of SEQ ID NOS: 57 and 58; The primers that complementarily bind to the D21S11 genetic locus are those of Sequence Listing 59 and Sequence 60; Wherein the primer complementarily binding to the Penta D genetic locus is a sequence selected from the group consisting of SEQ ID NOS: 61 and 62; The primers complementarily binding to the D3S1358 genetic locus are SEQ ID NO: 63 and SEQ ID NO: 64; Wherein the primers complementarily binding to the TH01 genetic locus are those of SEQ ID NOS: 65 and 66; Wherein the primers complementarily binding to the vWA genetic locus are SEQ ID NOS: 67 and 68; And
(b) determining an STR (short tandem repeat) allele of the genetic locus using the NGS data of the multiplex amplification product of step (a), and identifying the human object as a gene.
18. The method of claim 16, wherein the DNA sample is DNA isolated from a tissue selected from the group consisting of blood, semen, vaginal cells, hair, saliva, urine, oral cells, placental cells or amniotic fluid including fetal cells, Wherein the sample is a sample.
17. The method according to claim 16, wherein the DNA sample is a biological sample containing DNA.
17. The method of claim 16, wherein the DNA sample is a degraded DNA sample.
17. The method of claim 16, wherein the multiplex gene amplification is Polymerase Chain Reaction (PCR) amplification or direct multiplex PCR amplification.
17. The method of claim 16, wherein the multiplex gene Wherein the amplification has 31-38 cycles.
20. The method of claim 19, wherein the multiplex gene Wherein the amplification has 34-38 cycles.
17. The method of claim 16, wherein the multiplex gene amplification has an annealing temperature condition of 57-61 &lt; 0 &gt; C.
delete 17. The method according to claim 16, wherein the primer complementarily binding to the D19S433 genetic locus of step (a) has a final concentration of 0.4-0.8 [mu] M and the primer that binds complementarily to the D5S818 genetic locus is 0.3-0.8 [mu] M Wherein the primer complementarily binding to the Penta E genetic locus has a final concentration of 0.8-1.2 μM and the primer complementarily binding to the CSF1 PO genetic locus has a final concentration of 0.3-0.7 μM , The primer complementarily binding to the D7S820 genetic locus had a final concentration of 0.3-0.7 [mu] M, the primer complementarily binding to the D18S51 genetic locus had a final concentration of 0.7-1.1 [mu] M and the TPOX locus The complementarily binding primers have a final concentration of 0.1-0.6 [mu] M; The primer that binds complementarily to the D16S539 genetic locus has a final concentration of 0.2-0.7 [mu] M; The primer that binds complementarily to the D8S1179 genetic locus has a final concentration of 0.7-1.1 [mu] M; The primer that binds complementarily to the amelogenin gene locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the FGA gene locus has a final concentration of 0.8-1.2 [mu] M; The primer that binds complementarily to the D13S317 genetic locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the D2S1338 genetic locus has a final concentration of 0.6-1 [mu] M; The primer that binds complementarily to the D21S11 genetic locus has a final concentration of 0.5-0.9 [mu] M; The primer that binds complementarily to the Penta D genetic locus has a final concentration of 0.8-1.2 [mu] M; The primer that binds complementarily to the D3S1358 genetic locus has a final concentration of 0.3-0.7 [mu] M; The primer that binds complementarily to the TH01 genetic locus has a final concentration of 0.3-0.7 [mu] M; Wherein the primer complementarily binding to the vWA genetic locus has a final concentration of 0.8-1.2 [mu] M.
17. The method according to claim 16, wherein step (b) comprises comparing the nucleotide sequence of each STR allele amplified in the multiplex amplification product with a reference sequence, Lt; RTI ID = 0.0 &gt; STR &lt; / RTI &gt;
27. The method of claim 26, wherein the reference sequence comprises a sequence of the STR region of the allele, a 5 'perimeter sequence of the STR region, and a 3' perimeter sequence of the STR region.
27. The method of claim 26, wherein the method further comprises, after step (b), identifying the number of copies of the repeat sequence, the repeat structure of alleles, or sequence variation in the STR genomic locus RTI ID = 0.0 &gt; 1, &lt; / RTI &gt;
27. The method of claim 26, wherein the method further comprises the step of measuring the mixing ratio of the mixed sample using the sequence variation, wherein the mixing ratio is determined by analyzing coverage ratio between alleles on each STR gene, Lt; RTI ID = 0.0 &gt; mutation &lt; / RTI &gt; base sequence.

17. The method of claim 16, wherein the multiplex amplification product of step (b) has a size of 50 bp to 300 bp.
17. The method of claim 16, wherein the method of autosomal analysis has the use of forensic typing or identification.
17. The method of claim 16, wherein the primer is a non-labeled primer.
(Human c-fms proto-oncogene for CSF-1 receptor gene), D7S820, D18S51, TPOX (human thyroid peroxidase gene), D16S539, D8S1179, amelogenin, FGA (next generation sequencing-based) autosomal chromosomes containing respective primers complementarily binding to the genetic locus of the fibrinogen alpha chain, D13S317, D2S1338, D21S11, Penta D, D3S1358, TH01 and vWA (von Willebrand factor A) Wherein the primers complementarily binding to the D19S433 genetic locus are those of Sequence Listing 33 and Sequence 34; Wherein the primers complementarily binding to the D5S818 genetic locus are SEQ ID NO: 35 and SEQ ID NO: 36; Wherein the primer that binds complementarily to the Penta E gene locus is a sequence of SEQ ID NOS: 37 and 38; The primers complementarily binding to the CSF1PO genetic locus are SEQ ID NO: 39 and SEQ ID NO: 40; The primers complementarily binding to the D7S820 genetic locus are those of Sequence Listing 41 and Sequence 42; The primers complementarily binding to the D18S51 genetic locus are those of Sequence Listing 43 and Sequence 44; Wherein the primers complementarily binding to the TPOX genetic locus are those of Sequence Listing 45 and 46; The primers complementarily binding to the D16S539 genetic locus are those of Sequence Listing 47 and Sequence 48; The primers complementarily binding to the D8S1179 genetic locus are those of Sequence Listing 49 and Sequence 50; Wherein the primers complementarily binding to the amelogenin gene locus are SEQ ID NO: 51 and SEQ ID NO: 52; Wherein the primers complementarily binding to the FGA gene locus are SEQ ID NOS: 53 and 54, and the primers complementarily binding to the D13S317 genetic locus are SEQ ID NOS: 55 and 56; The primers complementarily binding to the D2S1338 genetic locus are those of SEQ ID NOS: 57 and 58; The primers that complementarily bind to the D21S11 genetic locus are those of Sequence Listing 59 and Sequence 60; Wherein the primer complementarily binding to the Penta D genetic locus is a sequence selected from the group consisting of SEQ ID NOS: 61 and 62; The primers complementarily binding to the D3S1358 genetic locus are SEQ ID NO: 63 and SEQ ID NO: 64; Wherein the primers complementarily binding to the TH01 genetic locus are those of SEQ ID NOS: 65 and 66; Wherein the primers complementarily binding to the vWA genetic locus are SEQ ID NOS: 67 and 68.

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