KR101913735B1 - Internal control substance searching for inter­sample cross­contamination of next­generation sequencing samples - Google Patents

Abstract

The present invention relates to a recombination vector containing oligonucleotide for searching cross-contamination between next generation sequence samples, and a next generation sequence analysis method capable of searching cross-contamination between the next-generation sequence analysis samples by using the recombination vector. The oligonucleotide has one or more sequences selected from the group consisting of the sequences of SEQ ID NOs: 1 to 21.

Description

An internal control substance for inter-sample cross-contamination search for next-generation sequence sequencing has been proposed for cross-contamination of nextgeneration sequencing samples.

The present invention relates to a recombinant vector containing an oligonucleotide for cross-contamination detection of a next-generation nucleotide sequence analyzing sample, and a next-generation sequencing method capable of searching for cross-contamination between next-generation nucleotide sequence analysis samples using the recombinant vector.

A variety of biometric information is expressed as genes of the DNA sequence, and the complete DNA sequence information of the individual is very important because it can understand the life phenomenon and obtain information related to the disease. At the core of genome sequencing is the identification of genetic sequencing, identification of individual differences and genetic characteristics, identification of genetic defects including chromosomal abnormalities in diseases associated with gene abnormalities, and genetic defects in complex diseases such as diabetes and hypertension . In addition, sequencing data is very important because it can widely utilize information such as gene expression, genetic diversity, and its interaction in molecular diagnostics and therapy.

Since 2007, Next Generation Sequencing (NGS) has been used as a method for genome sequencing, and as NGS has been developed, it has become much easier and less costly to analyze than traditional methods. Roche / 454, Illumina / Solexa, and SOLiD of Life Technologies (ABI) are examples of next generation sequencers that implement next-generation sequencing. These next-generation sequencing instruments can read more than 80 million sequences in 7 hours. These advances in technology have made it possible to utilize the next-generation sequencing method, which was previously used only for research purposes, in medical clinical tests due to the huge cost of testing.

However, when using the existing method of next generation nucleotide sequencing of Illumina, there is a problem in that cross-contamination between samples that can occur before attaching an index sequence including identification information for each sample can not be separated at the time of sample preparation.

Accordingly, in the present invention, an attempt was made to confirm whether the cross-contamination occurred through the aerosol and the error of the experimenter before the index sequence was attached by mixing an internal test substance having a bar code sequence that can identify each sample at the time of preparing the initial sample.

One aspect provides a recombinant vector comprising oligonucleotides for cross-contamination detection of next generation sequencing assays.

Another aspect provides a next generation sequencing method capable of detecting cross-contamination between next-generation sequencing assays using the recombinant vectors.

One aspect provides a recombinant vector comprising oligonucleotides for cross-contamination detection of next generation sequencing assays.

Next generation sequencing (NGS) is a method of quickly deciphering vast quantities of genomic information by disassembling a myriad of fragments, reading each fragment in supra-parallel, and combining it with computational techniques. The next generation sequencing method can generate a large amount of nucleotide sequence data for a sample to be analyzed within a short time.

In the present invention, super parallel sequencing includes a method known as a next generation sequencing method and a method that can be developed in the future. The superparallel sequencing can be performed by sequencing by synthesis, ion torrent sequencing, pyrosequencing, sequencing by ligation, nanopore sequencing, single-molecule real-time sequencing, polynucleotide sequencing, polymorphic sequencing, massively parallel signature sequencing (MPSS), DNA nanoball sequencing, and Heliscope single molecule sequencing.

Next-generation sequencing is used in a variety of applications including, for example, whole genome sequencing, which analyzes the entire genome of an individual, and exome analysis, Sequencing (exome sequencing), targeted sequencing for analyzing only the targeted region of the entire genome sequence, transcriptome sequencing for analyzing the nucleotide sequence of all the expressed RNAs, DNA methylation, An epigenome for analyzing histone deformation and non-coding RNA, and a metagenome for analyzing a microorganism community. The field of using next-generation sequencing has continued to expand.

A recombinant vector comprising an oligonucleotide for NGS inter-species cross-contamination search of the present invention is available in any of the above-described sequencing methods and in any field.

The next-generation sequencing method can be divided into two stages, depending on the sequencing method and field. The first step is the preparation of an NGS library to make the DNA to be analyzed into a form that can be sequenced by a next-generation sequencing analyzer. The second step is to analyze the prepared NGS library with a next-generation sequencing analyzer.

Generally, the steps of preparing the DNA library include the following.

(1) a sample preparation step of extracting genomic DNA from an analyte;

(2) fragmenting the extracted genomic DNA;

(3) End repair and adenosine conjugation (dA-Tailing): Two single-stranded DNAs constituting the fragmented double-stranded DNA are aligned with each other at the same length, and a base A is bonded to the 3'- do.

(4) Adapter bonding: Adapts the adapter to double-stranded DNA fragments for sequencing by next-generation sequencing instruments. This adapter may contain an index sequence capable of labeling each sample.

(5) Polymerase chain reaction (PCR): The DNA to be analyzed is amplified by PCR. At this time, by performing PCR using an index primer in which an index base sequence is inserted, index base sequence introduction and amplification can be performed on the DNA to be analyzed.

The pool of DNA molecules generated through the above process is referred to as an " NGS library ".

It is difficult to separate cross-contamination between samples that can occur before attaching the index sequence including identification information for each sample in the (4) adapter splicing step or (5) PCR step in the production step of the NGS library. In the present invention, the internal test substance having a bar code sequence capable of identifying each sample in the above (1) sample preparation step is introduced so that cross-contamination between samples can be confirmed before the adapter bonding and substantially at all stages of the next generation nucleotide sequence analysis have.

In the present invention, the NGS cross-contamination oligonucleotide and the recombinant vector containing the NGS sample are used to search for cross-contamination between NGS samples and to distinguish different samples from each other. Due to this feature, the oligonucleotide and the recombinant vector comprising it can be used interchangeably with the term 'barcode', 'spike-in' or 'internal test substance'.

The term 'barcode' refers to a unique sequence of nucleotides that can be used to distinguish each different sample collected into one NGS library.

The term 'internal test substance' refers to a substance used for the purpose of confirming whether the previous step of the next-generation sequencing method has proceeded by inserting a non-target sequence into the same sample container and amplifying it simultaneously with the target sequence (that is, Quot; non-target base sequence ").

In the present invention, a barcode sequence which is an oligonucleotide for NGS cross-contamination search is introduced into a sample in the form of (1) a sample inserted into a vector, and the index sequence used in conventional NGS is the Adapter bonding step, or (5) PCR step. This allows you to determine whether cross-contamination can occur before attaching the index sequence.

In the present invention, any sequence can be used as an oligonucleotide for cross-contamination detection of the NGS samples of the present invention, as long as it can serve as a bar code sequence, 'Next Generation Sequence Analysis (NGS) Cross-contamination search oligonucleotide'. Specifically, such oligonucleotides may be of length 2 to 1000, 3 to 100, 4, 5, 6, 7, 8, 9, 10, 11, To 50, 5 to 20 oligonucleotides. More specifically, the oligonucleotide may have one or more sequences selected from the group consisting of the sequences of SEQ ID NOS: 1 to 21.

The oligosaccharides for cross-contamination detection of NGS of the present invention can be used in one or a combination of two or more so that a large number of samples can be distinguished. Specifically, the oligonucleotide sequences 21 can be divided into a plurality of 21 or more samples by combining two or more oligonucleotides. For example, sample 1 uses the oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 as bar codes, sample 2 uses bar codes as oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 3, and bar code combination of sample 1 and sample 2 It can be distinguished later.

When the oligonucleotides are used in combination, each of the oligonucleotides may be used in a form inserted into a separate vector, or two or more oligonucleotides may be inserted into one vector.

The oligonucleotides or combinations thereof may be introduced into the NGS sample in the form embedded in a vector. In the present invention, the term 'vector' refers to a DNA molecule capable of replication and transferring a foreign DNA such as a gene to a recipient cell, such as a plasmid, a phage, a cosmid or an artificial chromosome.

In the present invention, the vector is not particularly limited as long as it is capable of delivering the oligosaccharide for NGS cross-contamination detection to the NGS sample, and any vector known in the art can be used. Examples of commonly used vectors include plasmids, cosmids, viruses and bacteriophages in their natural or recombinant state.

Specifically, a vector consisting of a nucleotide sequence that does not interfere with the analysis of an individual (for example, a human) to be analyzed due to the similarity of the nucleotide sequence or the like, or a vector modified according to the purpose can be used.

In the present invention, for example, pWE15, M13, lambda BL3, lambda BL4, lambda lII, lambda AS11, lambda ApII, lambda tlO, lambda tl l, Charon4A and Charon21A can be used as a phage vector or cosmid vector. , pBluescriptII system, pGEM system, pTZ system, pCL system, pET system and the like can be used. Specifically, a pUC-based vector, more specifically, pUC18 or pUC19 vector can be used. Alternatively, a vector derived from a pUC18 vector having high sequence homology with pUC18 may be used, or a vector derived from a pUC19 vector having high sequence homology with pUC19 may be used.

The use of pUC18 or pUC19 vectors does not affect the human genome data used in most of the next generation sequencing assays and has the advantage of being easy to mass-produce as high-copy plasmids. In addition, it is possible to carry out a general next-generation sequencing analysis method after mixing with a sample to be analyzed without going through a separate pretreatment process.

An oligonucleotide for cross-contamination detection of NGS samples is inserted into the vector to construct a recombinant vector. The term 'recombination' refers to the fact that elements such as DNA or RNA, which constitute genes, are changed from the original sequence during disassembly and reassembly.

The recombinant vector containing the oligonucleotide can be prepared according to a conventional method of linking two or more nucleic acid fragments. Specifically, it can be manufactured using the Gibson assembly method (FIG. 1). For example, a commercially available Gibson assembly kit can be used. When using this kit, PCR amplification is performed so that the fragments to be assembled overlap each other, and the amplified PCR fragments are mixed with a Gibson assembly master mix and reacted at about 50 ° C for 15 to 240 minutes. Then, a single-stranded overhang is created by the action of T5 exonuclease, and after annealing each of the overhangs, the DNA polymerase is filled up to the deleted base and extended to 3 ' , A reaction occurs in which the DNA ligase restores Nick, and a recombination vector containing oligonucleotides for the desired cross-contamination detection of NGS samples can be produced.

The recombinant vector thus produced may be transformed into an appropriate host and produced in large quantities. As the host, a host having high transformation efficiency and easy cultivation can be used. Specifically, it may be E. coli, for example E. coli HB101, JM109, DH5 ?, CJ236, BMH71-18 mutS, MV1184, TH2 which are competent cells with enhanced transformation efficiency. Transformation can be performed by a method commonly used in the art, and an efficient transformation method can be used for the selected host. For example, there are the CaCl ₂ precipitation method, the Hanahan method which increases the efficiency by using DMSO (dimethyl sulfoxide) in the CaCl ₂ method, and the electroporation method.

The transformed host can be cultured under culture medium and culture medium suitable for the selected host. The microorganism can be grown in a conventional medium, for example, in a nutrient broth medium. The culture medium may contain nutrients required for culturing, that is, a microorganism to be cultured in order to cultivate a specific microorganism, and may be a mixture in which a substance for a special purpose is further added and mixed. The recombinant vector transformed with the host population as well as the culture can be amplified and the desired recombinant vector can be isolated from the thus cultured culture.

Another aspect provides a next-generation sequencing method that can detect cross-contamination between samples using a recombinant vector containing oligonucleotides for cross-contamination search.

In the next generation nucleotide sequence analysis, the oligonucleotides for NGS cross-contamination detection and the recombinant vectors containing them are as described above. In addition, the next-generation sequencing method can introduce any of the sequencing methods described above and can be used in any application.

In the present invention, as described above, in the first step of the next-generation sequencing method (1), the DNA to be analyzed and the recombination vector containing the oligonucleotide for cross-pollination between the NGS samples are mixed ). That is, the recombinant vector can be added together with the DNA to be analyzed inserted into the container (e.g., tube). Here, the mixing ratio of the DNA to be analyzed and the recombinant vector may be 1: 0.1 - 1000, 1: 0.5 - 500, 1: 1 - 300, 1: Generally, it is easy to determine whether cross-contamination occurs when the recombinant vector is added excessively, but analysis noise may occur when the recombination vector is excessively excessive.

Next, the mixture is subjected to sample preparation for general sequencing by sequencing fragmentation (FIG. 3). Considering that the length of the DNA fragments read for each target NGS differs, if the size of the DNA sample is large, it is possible to apply the process of splicing to an appropriate size as required. The DNA fragmentation can be accomplished using physical means such as acoustic shearing, ultrasonic treatment, or hydrodynamic shearing. In addition, enzymatic means such as DNase I or other restriction enzymes or non-specific nucleases or transposase enzymes can be used for genomic DNA fragmentation .

Acoustic shearing and sonication are the major physical methods used to fragment DNA and can be performed using commercially available tools. For example, the Covaris tool (Woburn, Mass.) Is an audio instrument that can make DNA fragments in the size range of 100 bp to 5 kb. Bioruptor (Denville, NJ) is an ultrasonication device suitable for shearing chromatin and DNA to make genome fragments up to 1 kb in length. Hydroshear from Digilab (Marlborough, Mass.) Uses hydrodynamic forces for DNA shear. Nebulizers (Life Tech, Grand Island, NY) can also be used to atomize using compressed air, which allows DNA to be sheared into 100 bp - 3 kb fragments in a matter of seconds.

After the fragmentation, the conventional NGS library preparation process such as end repair and adenosine splicing, adapter splicing, and PCR is performed as described above. In the sequencing step, the sequence of the oligonucleotide for cross-pollination search between the NGS samples according to the present invention can be checked to determine whether there is cross-contamination between the samples in the next step of the next-generation nucleotide sequence analysis.

When the next generation sequencing method of the present invention is aimed at target sequencing, it may further include a target capture step of separating only genomic DNA regions to be analyzed from the NGS library before sequencing. Here, the NGS library is prepared by mixing a DNA to be analyzed and a recombination vector containing an oligonucleotide for cross-contamination search between NGS samples of the present invention in a sample preparation step.

The term 'target sequencing' is a typical method for identifying variants of various genes by capturing and analyzing only the targeted regions of the genome, rather than the entire genomic DNA.

The term " target capture " is a method for isolating and / or increasing the frequency of a particular gene or other region of interest from a DNA library prior to sequencing, keeping the region of interest for sequencing and removing the remaining material. Target capture methods include in-solution capture, hybridization capture, MIP (Molecular Inversion Probe) capture, and multiplexing PCR.

In this case, in the target capture step, a probe capturing an area to be analyzed in the genomic DNA and a probe capturing an oligonucleotide region for cross-contamination search between NGS samples, which have been mixed in the sample preparation step, Region and an oligonucleotide region (Fig. 3). The mixing ratio of the probes may be 1: 0.1 - 1000, 1: 0.5 - 1000, 1: 1 - 500, 1: 1 - 200. Here, when a probe for capturing the oligonucleotide region is mixed at a high rate, it is easy to judge contamination, but there is a disadvantage that the ratio of noise also increases. When the probe is mixed at a low ratio, such a disadvantage can be overcome and the barcode can be completely sequenced And thus can be economical.

The probe used herein may be, for example, 75 to 200 nucleotides, 80 to 200 nucleotides, 90 to 200 nucleotides, 100 nucleotides, or 100 nucleotides, each of which has a nucleotide sequence capable of specifically hybridizing with a part of the region 100 to 180, 100 to 160, 100 to 140, and 100 to 120 nucleotides. If the probe has a size of 75 or less, the capturing accuracy for the target area is low. If the size of the probe is 200 or more, the synthesis cost is increased.

The probe may be a set of a plurality of probes, and the probe set may be fabricated by a tiling technique. That is, any probe constituting the set and another probe including the nucleotide sequence of the nearest region to be analyzed may be made to have the same sequence. In this case, each of the probe sequences constituting the set includes a part of the region sequence to be analyzed, and the sequence of the region to be analyzed which is not included in the probe may not exist. This means that the entire sequence of the region to be analyzed can be covered by the probes constituting the set.

In the case of the probe set prepared by the tiling technique, one nucleotide of the nucleotide sequence of the region to be analyzed can be covered by two or more kinds of probes, specifically three or more kinds of probes.

In this case, the arbitrary probe constituting the probe set and the other probe closest to the probe set may be, for example, 50 to 150, 60 to 140, 70 to 120, 70 to 110, 70 to 100, 70 To 90, and 70 to 80 identical sequences.

The probe may be prepared from DNA, RNA, Peptide Nucleic Acid (PNA), Locked Nucleic Acid (LNA), Zip Nucleic Acid (ZNA), Bridged Nucleic Acid (BNA) It can be one or more selected. The probe may specifically be DNA or RNA, more specifically RNA.

The probes can be synthesized using any method known to be capable of synthesizing oligonucleotides in the art, for example, using an automated DNA synthesizer (such as those available as Biosearch, Applied Biosystems ^TM, etc.) . The probe may be transcribed to generate an RNA polynucleotide that specifically hybridizes to an area to be analyzed. The transcription may be in-vitro transcription.

The probe may further comprise a moiety for its isolation or purification. The moiety may be attached to one or more of the nucleotides constituting the probe. The moiety may include one or more selected from the group consisting of biotin, avidin, and streptavidin. Further, the moiety, for example, biotin, avidin or streptavidin, may include a magnetic bead, or a substance that specifically binds to the moiety may include magnetic beads. The separation or purification may be carried out by a substance or a magnetic field that specifically binds to the moiety.

In the present invention, the probe may be designed to further design a terminal probe starting from both ends of the region to be analyzed in order to improve on-target ratio and uniformity of the probe. Since the capture efficiency may be lowered when the probe has a high GC ratio at a position where the probe binds to the target region or when a specific sequence repeatedly appears, a method of adjusting the probe sequence (when four or more G or C are continuous A GC fix process in which the fourth base is substituted with A or T) is applied to the first and second probes.

The probe that captures the oligonucleotide region for cross-contamination detection between the NGS samples may be made by a tiling technique for efficient capture. In the present invention, the probe can be fabricated, for example, by a 3 x tiling technique (FIG. 2).

The oligonucleotide sequence thus captured can be identified in the sequencing step to determine whether there is cross-contamination between samples at the entire NGS stage, including sample preparation.

During next-generation sequencing, sample cross-contamination that can occur before attaching the adapter can be detected during sample preparation.

The internal test substance of the present invention is simple and enables cross-contamination between samples without affecting analytical specimens.

The internal test material of the present invention is in the form of a recombinant vector, and it is advantageous that all the processes of NGS including the DNA fragmentation process can be treated in the same manner with the DNA sample to be analyzed without a complex pretreatment process after production.

Figure 1 shows the process for constructing a pUC19 vector containing oligonucleotides for cross-contamination detection of NGS samples.
FIG. 2 shows a process for producing a probe capable of capturing an oligonucleotide region for NGS cross-contamination detection.
FIG. 3 shows a procedure for introducing a hybridization capture probe necessary for mixing and detection of a recombination vector containing oligonucleotides for NGS cross-contamination search in a sample preparation process performed in the next generation nucleotide sequence analysis.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these embodiments are provided to aid understanding of the present invention, and the scope of the present invention is not limited thereto in any sense.

Example 1: Design and manufacture of internal test substances

Based on the pUC19 vector, an internal test substance containing a barcode was designed. When pUC19 vector is used, it is used in most of next-generation sequencing methods and does not affect human genome data. It is advantageous in mass production as a high-copy plasmid. Therefore, the sample preparation for the next generation sequencing can be performed by mixing the inner test substance prepared in the plasmid form to the gDNA of the sample to be analyzed without any separate pretreatment, and proceeding with shearing.

As shown in Table 1, 21 kinds of oligonucleotides having a length of 10 bp were prepared as barcode sequences to be inserted into the pUC19 vector. Then, as shown in Fig. 1, the pUC19 vector was treated with a restriction enzyme, and then a pUC19 vector having a bar code was prepared using the Gibson assembly method.

Bar code name Base sequence SEQ ID NO: spike in_1 GATTGATGCC One spike in_2 CTGGCGTCGG 2 spike in_3 GACTATGCGA 3 spike in_4 CGCAGCGTAA 4 spike in_5 AGGTCGCGCG 5 spike in_6 GATTGCACAG 6 spike in_7 AAGATCTCGT 7 spike in_8 GGCATTGCTG 8 spike in_9 CTGTCTCGTT 9 spike in_10 ATTCTCCACC 10 spike in_11 TCCGGTAGTA 11 spike in_12 GAACTGGATG 12 spike in_13 ATAGCAGGTG 13 spike in_14 GATCGCTTGG 14 spike in_15 AGCTACTAGT 15 spike in_16 AGTAGTTATT 16 spike in_17 ACTCTTCTGG 17 spike in_18 AATCCGCGTT 18 spike in_19 ATCTGCACAT 19 spike in_20 AGTATTGAGA 20 spike in_21 AGTCGGCAGT 21

Twenty-one internal test substances can not be distinguished as bar codes for only 21 samples, but more than 21 samples can be separated into bar codes by a combination of two or more internal test substances. For example, sample 1 uses spike in_1 and spike in_2 as bar codes, and sample 2 uses bar codes as spike in_1 and spike in_3, so that the combination of sample 1 and sample 2 has different bar codes.

Example 2: Design of a next-generation sequencing method using an internal test substance

After the internal test substance was mixed with the next-generation sequencing target sample, the next-generation sequence sequencing was performed. Thereafter, a probe for capturing a target region of the human genome and a probe capable of capturing a bar code region produced by the 3 x tiling technique as shown in FIG. 2 were carried out in hybridization capturing performed in preparation for target sequencing . The three probe sequences capable of capturing the internal test substance are as follows.

The N portion included in the following sequence corresponds to the bar code (where N is one of A, G, C, and T bases) and can be used regardless of the type of the bar code by adjusting this portion according to the bar code sequence.

5 'GGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTNNNNNNNNNAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCT-3' (SEQ ID NO: 22)

5 'CAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTNNNNNNNNNNAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCT-3' (SEQ ID NO: 23)

5 'TTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTNNNNNNNNNNAATTCGAGCTCGGTACCCGGGGATCCTCTAGAGTCGACCTGCAGGCATGCAAGCTTGGCGTAATCATGGT -3' (SEQ ID NO: 24)

During the final analysis, the barcode sequence of the captured internal test substance was identified, and it was possible to judge whether cross contamination occurred between the samples during the sample preparation process.

FIG. 3 shows a procedure for introducing a hybridization capture probe necessary for mixing and detection of internal test substances in the sample preparation process performed in the conventional next-generation sequencing.

Example 3: Experimental investigation of actual cross-contamination using an internal test substance

3.1: Experiments without cross-contamination

Experiments were conducted to determine whether cross-contamination between samples could be detected during the preparation of next-generation sequencing analysis samples using the internal test materials prepared in the above examples. NA12878 was mixed with NA12878 at a ratio of 1: 100 on a mole number basis. At this time, as shown in Table 2, the internal test substance was inserted into two or more bar codes.

Sample number Sample Index Internal test substance bar code One 8 10, 11 2 9 12, 13 3 15 6, 7 4 16 18, 19

The target hybridization capture was proceeded with the BRCA kit. In this process, the probe capable of capturing the internal test substance was mixed with the BRCA hybridization capture probe at a ratio of 1: 1, and hybridization Capture.

Table 3 shows the experimental conditions and the frequency of detection of the internal test substances in the next generation sequencing using the final test product. Bar codes that should not have been detected in sample 1 and sample 3 were detected at the frequency of bar code detection for each sample index, but the detection amount was very small and it was judged to be analytical noise. In the other samples, only the barcode of the originally mixed internal test substance was normally detected.

3.2. Experiments in the presence of cross-contamination

The presence of forced cross-contamination under the same conditions as in Example 3.1 above, and then whether cross-contamination can be detected can be confirmed through the bar code mixed state of the internal test substance. Forced cross-contamination between Sample 3 and Sample 4 between Sample 1 and Sample 2 was confirmed and the internal test substance bar code was confirmed through sequencing analysis of the next generation.

Cross-contamination between samples was confirmed by examining the internal test substance barcode between sample 1 and sample 2 (Table 4). It can be seen that the frequency of the barcode to be displayed as the contamination increased sharply when compared with the result of the example in which the cross contamination was not generated artificially, and it was confirmed that the ratio also increased. This tendency was confirmed by the cross-contamination test between sample 3 and sample 4 (Table 4).

Example 4: Sample analysis by using internal test substance Investigation of contamination possibility

The internal test material is composed of the pUC19 vector, and it is searched whether it can be misinterpreted due to the nucleotide sequence similarity with the human genome to be sequenced. Internal sequencing was performed on the internal test material and the resulting data was aligned with the human reference genome. As a result, it was confirmed that a very low proportion of the internal test substance sequence was aligned with the human reference genome.

This means that considering the mixture ratio of the human genome and the internal test substance, the effect on the data analysis of the human genome in real data is negligible (Table 5). Also, from the results in Table 5, it can be seen that applying the Spike-in DNA to the vector and applying it to the NGS can also verify the cross-contamination of the sample without affecting the human genome data analysis.

In the in-silico analysis, it was confirmed that the spike-in vector did not have a sequence mapped to the human genome. In the actual NGS data, it was confirmed that an extremely low ratio of DNA was mapped to the human genome. In fact, when NGS is performed using spike-in, the amount of data derived from spike-in is very low depending on the mixing ratio of DNA, so the effect on human genome data is minimal.

<110> Celemics, Inc. <120> Internal control substance searching for inter-sample          cross-contamination of next-generation sequencing samples <130> SDP2018-1006 <160> 24 <170> KoPatentin 3.0 <210> 1 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 1 gattgatgcc 10 <210> 2 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 2 ctggcgtcgg 10 <210> 3 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 3 gactatgcga 10 <210> 4 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 4 cgcagcgtaa 10 <210> 5 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 5 aggtcgcgcg 10 <210> 6 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 6 gattgcacag 10 <210> 7 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 7 aagatctcgt 10 <210> 8 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 8 ggcattgctg 10 <210> 9 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 9 ctgtctcgtt 10 <210> 10 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 10 attctccacc 10 <210> 11 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 11 tccggtagta 10 <210> 12 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 12 gaactggatg 10 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 13 atagcaggtg 10 <210> 14 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 14 gatcgcttgg 10 <210> 15 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 15 agctactagt 10 <210> 16 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 16 agtagttatt 10 <210> 17 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 17 actcttctgg 10 <210> 18 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 18 aatccgcgtt 10 <210> 19 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 19 atctgcacat 10 <210> 20 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 20 agtattgaga 10 <210> 21 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> barcode <400> 21 agtcggcagt 10 <210> 22 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> probe for barcode <400> 22 gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt gaattnnnnn 60 nnnnnaattc gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct 120                                                                          120 <210> 23 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> probe for barcode <400> 23 caaggcgatt aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg 60 ccagtgaatt nnnnnnnnnn aattcgagct cggtacccgg ggatcctcta gagtcgacct 120                                                                          120 <210> 24 <211> 120 <212> DNA <213> Artificial Sequence <220> <223> probe for barcode <400> 24 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt nnnnnnnnnn aattcgagct 60 cggtacccgg ggatcctcta gagtcgacct gcaggcatgc aagcttggcg taatcatggt 120                                                                          120

Claims (11)

Next Generation Sequencing (NGS) Includes oligonucleotides for inter-sample cross-contamination detection,
Wherein the oligonucleotide has one or more sequences selected from the group consisting of the sequences of SEQ ID NOS: 1 to 21 and is contained as a barcode in a pUC-based vector treated with restriction enzyme using the Gibson assembly method. The recombinant vector of claim 1, wherein the Gibson assembly method utilizes a Gibson assembly kit. (B) an oligonucleotide having the sequence of SEQ ID NO: 12; and (c) an oligonucleotide having the sequence of SEQ ID NO: 12 and the oligonucleotide having the sequence of SEQ ID NO: (C) a combination of an oligonucleotide having the sequence of SEQ ID NO: 6 and an oligonucleotide having the sequence of SEQ ID NO: 7, (d) a combination of the oligonucleotide having the sequence of SEQ ID NO: 18 and the oligonucleotide having the sequence of SEQ ID NO: A combination of oligonucleotides having a sequence, and a combination of oligonucleotides having a sequence. The recombinant vector according to claim 1, wherein the pUC family vector is pUC18 or pUC19. Next Generation Sequencing Analysis Method Using the Recombinant Vector of Claim 1 Next Generation Sequence Analysis Method to Search for Cross-contamination between Samples. In claim 5, in the sample preparation step, a recombination vector containing the oligonucleotide for cross-pollination between the DNA to be analyzed and the NGS sample is mixed with each sample, and the oligonucleotide sequence is confirmed in the sequencing step, Wherein the method comprises the steps of: [Claim 6] The method according to claim 6, wherein the recombinant vector containing the oligonucleotide for cross-contamination between the DNA to be analyzed and the NGS sample is mixed at a ratio of 1: 0.1-1000. The method according to claim 5, wherein the next generation sequencing method is for target sequencing. In claim 8, in the sample preparation step, a recombination vector containing the oligonucleotide for cross-pollination between the DNA to be analyzed and the NGS sample is mixed with each sample, and in the target capture step, a probe And a method for capturing an oligonucleotide region and an oligonucleotide region to be analyzed using a probe for capturing the oligonucleotide region, and determining the sequence of the oligonucleotide region in the sequencing step to determine whether cross contamination between the samples is present. [Claim 9] The method according to claim 9, wherein the probe capturing the region to be analyzed and the probe capturing the oligonucleotide region among the genomic DNA are mixed at a ratio of 1: 0.1-1000. 6. The method of claim 6, wherein the sequencing is performed by sequencing by synthesis, ion torrent sequencing, pyrosequencing, sequencing by ligation, nanopore sequencing, single- Generation base, which is selected from the group consisting of polony sequencing, massively parallel signature sequencing (MPSS), DNA nanoball sequencing and Heliscope single molecule sequencing, Sequencing method.

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