Method and apparatus for generating SEED sequence for internal tandem repeat analysis in next generation sequencing analysis

Abstract

One embodiment of the present invention relates to a method for obtaining information relating to a read of an arbitrary sequence by a next generation sequencing (NGS) analysis method, and then screening a read having the same insertion sequence and a read having the same soft-clipped base sequence from among the read obtained above on the basis of a reference sequence. In addition, some or all of the parts containing the soft trimming base sequence and the insertion sequence that have been screened and read are selected as SEEDs, and the internal tandem duplication (ITD) is accurately analyzed by the selected SEED, whereby it is possible to diagnose, prognostically diagnose and the like diseases associated with the internal tandem duplication.

Description

Technical Field

[0001] Embodiments disclosed in the present invention relate to a method and apparatus for generating a SEED sequence for deriving internal tandem duplications (ITDs) in next-generation sequencing (NGS) analysis, and more specifically, to a method and apparatus for screening SEED sequences in order to conveniently distinguish ITDs from read sequences derived from NGS analysis. Background Art

[0002] Currently, NGS tests for diagnosing genetic diseases are being performed in medical settings around the world, and research in the field of precision medicine is being actively conducted through this. NGS technologies used in precision medicine include panel sequencing, exome sequencing, and whole genome sequencing. Although NGS can be used to sequence genes quickly and accurately, when analyzing internal tandem duplications (ITDs) using NGS, it is difficult to accurately analyze ITDs due to the limitations of NGS analysis.

[0003] Although various commercial analysis programs have been introduced in NGS analysis to solve the problems of ITD analysis, limitations are still shown in ITD analysis. The present invention is invented to solve the problems of commercial analysis software. Summary of the invention

[0004] Technical issues

[0005] In order to analyze ITD quickly and accurately, the embodiments disclosed in the present invention provide a method and apparatus for deriving a SEED for facilitating ITD analysis.

[0006] Technical Solution

[0007] Therefore, the present invention provides a method for deriving a sequence for analyzing internal tandem repeats in a next-generation sequencing method, characterized in that it comprises: step 1), obtaining reads by a next-generation sequencing method; step 2), a) screening reads having the same insertion sequence in the above-obtained reads based on a reference sequence, and / or b) screening reads having the same soft-clipped bases; and step 3), selecting a portion or all of the soft-clipped base sequence and / or insertion sequence of the above-screened reads as a SEED sequence (SEED).

[0008] According to an embodiment of the present invention, it is characterized in that in the step of screening reads in the above step 2), if three or more reads have the same insertion sequence, the above reads with the above same insertion sequence can be screened.

[0009] According to another embodiment of the present invention, it is characterized in that, in the step of screening reads in the above step 2), if more than three reads have the same soft-trimmed base sequence, the above reads with the above same soft-trimmed base sequence can be screened.

[0010] According to one embodiment of the present invention, it is characterized in that the portion containing the soft-trimmed base sequence in the above step 3) contains a sequence adjacent to the 3' or 5' end of the soft-trimmed base, and the sequence length of the above-mentioned sequence adjacent to the 3' or 5' end of the soft-trimmed base can be 12bp to 20bp.

[0011] According to another embodiment of the present invention, it is characterized in that the portion containing the insertion sequence in the above step 3) contains a sequence adjacent to the 3' or 5' end of the insertion sequence, and the sequence length of the above sequence adjacent to the 3' or 5' end of the insertion sequence can be 12bp to 20bp.

[0012] According to an embodiment of the present invention, the NGS method may be an amplicon-based NGS method.

[0013] In addition, another embodiment of the present invention discloses a method for analyzing ITD in an NGS method, comprising: step 1), obtaining reads by a next-generation sequencing method; step 2), a) screening the reads having the same insertion sequence in the above-obtained reads based on a reference sequence, and / or b) screening the reads having the same soft-trimmed bases; step 3), selecting a part or all of the soft-trimmed base sequence and / or insertion sequence of the above-screened reads as SEED; and step 4), querying the above-selected SEED, and analyzing the sequence matching the SEED for any read obtained by the next-generation sequencing method.

[0014] According to an embodiment of the present invention, the analysis in step 4) may be a step of counting the number of matched sequences.

[0015] Another embodiment of the present invention discloses an apparatus for deriving sequences for analyzing internal tandem duplications (ITD) in next-generation sequencing (NGS) analysis, a processor for obtaining information about reads of an arbitrary sequence through a next-generation sequencing method, screening reads having the same insertion sequence among the above-obtained reads based on a reference sequence, and / or screening reads having the same soft-trimmed bases, and selecting a portion or all of the soft-trimmed base sequence and / or insertion sequence of the above-screened reads as SEED; a memory for storing information about the above-mentioned reads, information about the reference sequence and SEED; and a display for displaying information about the above-mentioned derived SEED.

[0016] According to an embodiment of the present invention, it is characterized in that, in the step of screening the above-mentioned reads, if three or more reads have the same insertion sequence, the above-mentioned reads having the above-mentioned same insertion sequence can be screened.

[0017] According to another embodiment of the present invention, it is characterized in that, in the step of screening the above-mentioned readings, if more than three readings have the same soft-trimmed base sequence, the above-mentioned readings with the above-mentioned same soft-trimmed base sequence can be screened.

[0018] According to one embodiment of the present invention, it is characterized in that the portion containing the above-mentioned soft trimmed base sequence contains a sequence adjacent to the 3' or 5' end of the soft trimmed base, and the sequence length of the above-mentioned sequence adjacent to the 3' or 5' end of the soft trimmed base can be 12bp to 20bp.

[0019] According to another embodiment of the present invention, it is characterized in that the site containing the above-mentioned insertion sequence contains a sequence adjacent to the 3' or 5' end of the insertion sequence, and the sequence length of the above-mentioned sequence adjacent to the 3' or 5' end of the insertion sequence can be 12bp to 20bp.

[0020] Effects of the Invention

[0021] The method or apparatus of one embodiment of the present invention derives a SEED that can quickly and accurately perform specific ITD analysis in the reading obtained by NGS, so that the status and number of ITDs can be quickly and accurately derived from the NGS reading of the patient through the derived SEED. Therefore, the status of the disease suffered by the patient can be detected using the SEED. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 A conceptual diagram for explaining a method for deriving SEED according to an embodiment.

[0023] Figure 2 A diagram showing the effect of ITD analysis using SEED according to an embodiment.

[0024] Figure 3 A diagram showing an example of analyzing reads on an IGV using the SEED derived in the present invention.

[0025] Figure 4 FIG. 4 is a flowchart for illustrating a SEED derivation method according to an embodiment.

[0026] Figure 5 FIG. 4 is a flowchart for more specifically illustrating a SEED derivation method according to an embodiment.

[0027] Figure 6 A block diagram of an apparatus according to an embodiment. DETAILED DESCRIPTION

[0028] The terms used in this specification will be briefly described first, and then the present invention will be described in detail.

[0029] The terms used in the present invention are selected as common terms that are currently widely used as much as possible after considering the functions in the present invention, but these terms may be different according to the intentions of ordinary technicians in the technical field to which the present invention belongs, precedents, the emergence of new technologies, etc. In addition, in certain cases, there are terms that the applicant arbitrarily selects. In this case, their meanings will be described in detail in the relevant description. Therefore, the terms used in the present invention should not be defined only by the meaning of the simple term name, but should be defined based on the meaning of the term and the entire content of the present invention.

[0030] In the entire specification, when it is mentioned that a certain part "includes" a certain structural element, if there is no special record to the contrary, it means that other structural elements may also be included, rather than excluding other structural elements. In addition, the terms "... part", "module" and the like recorded in the specification mean a unit that processes at least one function or operation, which can be implemented by hardware or software or a combination of hardware and software.

[0031] The term "next generation base sequence analysis method" or "NGS" in the present invention refers to any sequencing method that determines a nucleotide sequence in an amplified proxy for each nucleic acid molecule (e.g., in single molecule sequencing) or by a high-throughput method (e.g., sequencing 10, 100, 1000 or more molecules simultaneously). Next generation sequencing methods are well known in the art to which the present invention belongs, for example, as described in Metzker, M. (2010) Nature Biotechnology Reviews 11: 31-46. Next generation sequencing can detect less than 5% variants present in nucleic acids in a sample.

[0032] The term "amplicon-based NGS method" of the present invention is a technique for aligning primers capable of amplifying target genes to produce short-length reads in a variety of ways for analysis. Representative techniques include the emulsion polymerase chain reaction (Emulsion PCR) method, based on which are Roche's 454 platform, Thermo Fisher's SOLid platform and Ion Torrent platform. Compared with the probe-based hybridization method, the NGS of the amplicon method has the advantages of low library complexity and high speed. The NGS data of the amplicon method has a primer sequence in the first part of the sequence read. The primer sequence is designed to be the same sequence as the standard sequence.

[0033] (1) Target selection

[0034] Usually, the method of sequencing the target is as follows. In order to find the causative gene of the disease, the whole-length genome (Whole-genome) can be sequenced using the next generation base sequence analysis method, or only the exon region can be sequenced (Targeted sequencing), or a specific gene can be targeted. In the case of sequencing only the exon region or a specific target gene, it is advantageous in terms of cost or efficiency. In addition, since the changes in genes mostly manifest as direct diseases such as cancer, it can be said that detecting changes in base sequences in the exon region or the target gene is effective in finding causative genes. In order to sequence only exons or target genes, a library that can capture only exons or target genes is needed.

[0035] (2) High-throughput parallel DNA sequencing

[0036] Compared with the existing capillary sequencing method, the next generation base sequence analysis method can quickly perform more sequence confirmation and can omit the process of using a vector to amplify the sample used in the existing capillary sequencing method, thus having the advantage of avoiding errors that occur in the process.

[0037] The NGS systems produced by three companies are mainly used. The 454GS FLX of Roche, mentioned above, was the first NGS device introduced in 2004. This device uses pyrosequencing and emulsion-polymerase chain reaction to confirm the sequence, and can confirm the specific base based on the intensity of light that appears in the final step of the experiment. It can confirm about 100Mb of sequence in 7 hours, which is significantly higher than the existing ABI 3730 instrument, which confirms 440kb of sequence in the same time.

[0038] Illumina's Illimina Genome Analyzer uses a concept called sequencing by synthesis, where a DNA fragment consisting of only one chain is attached to a glass plate and then clustered through a polymerization reaction. During this process, the sequence is analyzed by confirming the type of bases attached to the DNA fragment, and 40 to 50 million fragments with a length of 32 to 40 bases are produced in about 4 days.

[0039] The Oligonucleotide Ligation Sequencing (SOLiD) instrument of Life Technologies attaches the DNA fragment to be checked on a 1μm magnetic bead and then uses an emulsifier-enzyme chain reaction to confirm the sequence. When confirming the sequence, the 8-mer fragment is repeatedly attached. The bases to be used in the actual sequence confirmation are located at the fourth and fifth positions of the 8-mer fragment. The remaining sites attached are then connected with fluorescent substances to show which bases are complementary to the DNA fragment to be checked. 8-mer is attached 5 times in each binding cycle, and the same operation is performed 5 times, so that the sequence of the DNA fragment composed of 25 bases can be confirmed. The SOLiD instrument is characterized by sequence confirmation using two-base encoding. This method confirms the same site by two sequence confirmations when determining the sequence of one base. Sequence confirmation is performed by moving one base to the adaptor attached to the magnetic bead in each binding cycle. Therefore, there is an advantage that errors occurring in sequence confirmation experiments can be eliminated through this process.

[0040] (3) Analysis of base sequence data

[0041] In order to find the causative gene of the disease, it is necessary to investigate what changes have occurred in the existing gene base sequence, so it is necessary to compare the base sequence data (sequence reads) of an individual (patient) with the reference genome (reference Genome) or reference sequence (reference sequence). This operation is called mapping. After the difference between the individual and the reference genome is known through mapping, appropriate selection criteria are set to extract only reliable base sequence variation information (Variant Calling). This variation information includes structural variation (structural variation, SV) including single base sequence variation (SNV: Single Nucleotide Variation), short insertion / deletion (Short Indel), copy number variation (copy number variation, CNV) and fusion genes. Then, the base sequence variation pheromone is compared with the benchmark database to determine whether it is a variation that has been discovered or a newly discovered variation. In addition, it is also predicted whether the variation will cause changes in amino acids and what impact it will have on the protein structure. This process is called annotation. To further improve the quality of information, the extracted single base sequence variations and information on short insertions / deletions can be logged in to databases or integrated with genome-wide association studies (GWAS) to search for disease-causing variants.

[0042] The term "obtain" or "obtained" as used in the present invention refers to all meanings of "directly obtaining" a physical entity or value or "obtaining by indirect means" a physical entity or value, such as a numerical value, as used in this specification. "Directly obtaining" means performing a process (e.g., performing a synthesis or analysis method) for obtaining a physical entity or value. "Obtained by indirect means" means receiving a physical entity or value from another stakeholder or supply source (e.g., a third-party research institute that directly obtains the physical entity or value).

[0043] Indirect methods of obtaining a physical entity include methods of obtaining the physical entity, for example, methods including processing the starting material including physical changes. Representative changes include methods of preparing the physical entity from two or more starting materials, shearing or fragmenting the material, separating or purifying the material, combining two or more separate independent bodies into a mixture, and performing chemical reactions including breaking or forming covalent bonds or non-covalent bonds. Indirect ways of obtaining values ​​include methods of performing treatments including physical changes in samples or other substances, for example, performing analytical processes including physical changes in substances such as samples, analytes or reagents (sometimes referred to as "physical analysis" in this specification), performing analytical methods including, for example, the following methods: separating or purifying substances such as analytes, or fragments or other derivatives thereof from other substances; mixing analytes, or fragments or other derivatives thereof with other substances, such as buffers, solvents or reactants; or, changing the structure of an analyte, a fragment thereof, or other derivative by destroying or forming a covalent bond or non-covalent bond between, for example, a first atom of the analyte and a second atom thereof; or, changing the structure of a reagent, a fragment thereof, or other derivative by destroying or forming a covalent bond or non-covalent bond between, for example, a first atom of the reagent and a second atom thereof.

[0044] The term "obtaining a sequence" or "obtaining a read" in the present invention is used in this specification to refer to all of the nucleotide sequence or amino acid sequence obtained by "directly obtaining" or "indirectly obtaining". "Directly obtaining" a sequence or reading refers to a process (for example, performing a synthesis or analysis method) such as a sequencing method (for example, a next-generation sequencing (NGS) method) for obtaining a sequence. "Indirectly obtaining" a sequence or reading refers to receiving a sequence or receiving information or knowledge of a sequence from other stakeholders or supply sources (for example, a third-party research institute that directly obtains the sequence). The sequence or read obtained does not need to be a complete sequence. For example, obtaining the sequence includes obtaining sequencing of at least one nucleotide or obtaining information or knowledge of one or more of the changes disclosed in this specification for confirming the presence in the subject.

[0045] Directly obtaining a sequence or read includes performing a process including a physical change in a physical material, for example, in a starting material, such as a tissue or cell sample, such as a nucleic acid (e.g., DNA or RNA) sample obtained by biopsy or isolation. Representative changes are shearing or fragmentation of two or more starting materials, for example, including: preparing a physical entity from genomic DNA (e.g., isolating a nucleic acid sample from a tissue); combining two or more separate independent entities into a mixture; and performing a chemical reaction including the destruction or formation of covalent or non-covalent bonds. Directly obtaining a value includes performing a process including a physical change in a sample or other material as described above.

[0046] The term "nucleic acid" or "nucleotide" used in the present invention refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form and their polymers. If not particularly limited, the above term includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to standard nucleic acids and are metabolized in a manner similar to natural nucleotides. If not otherwise recorded, the specific nucleic acid sequence not only includes the sequence clearly recorded, but also includes variants (for example, degenerate codon replacements), alleles, orthologous genes, single nucleotide polymorphisms (SNPs) and complementary sequences of its conservative deformations by default. Specifically, degenerate codon replacement can be achieved by generating a sequence in which the 3rd position of more than one selected (or all) codons is replaced by mixed bases and / or deoxyinosine residues. The term "nucleic acid" mentioned above can be used interchangeably with gene, complementary deoxyribonucleic acid (cDNA), messenger ribonucleic acid (mRNA), small non-coding ribonucleic acid, microRNA (miRNA), Piwi-interacting ribonucleic acid or short hairpin ribonucleic acid (shRNA) encoded by a gene locus.

[0047] The term "paired-end read" in the present invention refers to the two ends of the same deoxyribonucleic acid molecule. When one end is sequenced and then flipped to sequence the other end, the two ends whose base sequences are ascertained are called "paired-end reads". For example, Illumina sequencing generates a read of about 500bps, and reads 75bps of base sequence at both ends of the read. In this case, the directions of reading the two reads (first read and second read) are 3' and 5', and the directions are opposite to each other, thereby forming a mutual double-end read.

[0048] The terms "soft-clip", "soft-clip segment" or "soft clipped read" in the present invention quantify the reads obtained from NGS in which only a portion is mapped to the reference genome (reference sequence), while the rest is unmapped.

[0049] The term "soft-clip base" used in the present invention refers to an unmatched sequence that exists after the end of the matched portion in a soft-clipped read after matching with a reference sequence.

[0050] The term "brick point" in the present invention refers to the end of a sequence of which only a portion is mapped to a reference genome (reference sequence) in a "soft-trimmed read".

[0051] The term "insertion sequence" in the present invention refers to a sequence that is additionally inserted in a read by comparison with a reference sequence.

[0052] The term "disconcordant read pair" in the present invention refers to a read pair (first read, second read) obtained by paired-end sequencing that is not mapped to a reference gene but is mapped to different positions or different chromosomes.

[0053] The term "concordant read pair" in the present invention refers to a read pair (first read, second read) obtained by double-end read sequencing that is mapped to the same gene, while the soft-trimmed fragment portion of the read has information mapped in other genes.

[0054] The term "SEED (SEED sequence)" in the present invention refers to a sequence derived in the present invention in order to rapidly and accurately perform ITD analysis.

[0055] Embodiments of the present invention

[0056] The present invention will be described in more detail below by way of examples. These examples are only used to more specifically describe the present invention, and the scope of the present invention is not limited to these examples.

[0057] According to one embodiment of the present invention, a method for deriving a SEED for rapidly and accurately performing an ITD analysis in an NGS analysis of a specific target sequence is provided.

[0058] Reference Figure 1In one embodiment, the method for exporting SEED can be to load the BAM file generated by the amplicon method into the Integrative Genomincs Viewer (IGV), set the maximum downsized read count to 10,000, and then align the reads by insertion size to confirm whether there is an insertion of the same sequence in more than three reads. Then, the reads can be aligned by base to confirm whether there is a soft-trimmed base of the same sequence in more than three reads. The confirmed sequence is used to determine the SEED of about 8 bp to 30 bp passing through the boundary of the insertion sequence or the soft-trimmed base sequence, preferably, the SEED of about 12 bp to 20 bp. Then, the samtool command is used to count the number of reads containing the determined SEED and divide it by the total count to determine the variant allele frequency (VAF).

[0059] Figure 2 The figure compares the results of ITD analysis using SEED derived from an embodiment with the results of ITD analysis using other methods. Specifically, simulations were performed using various methods based on 53 known NGS read information and ITD information.

[0060] like Figure 2 As shown, when analyzing all 53 ITDs, all ITDs were found by the method of the present invention, but only a part of them could be found by other methods.

[0061] Figure 3 This is an example of using SEED derived from an embodiment to perform ITD analysis.

[0062] Figure 4 FIG. 4 is a flowchart for illustrating a method for deriving SEED according to an embodiment.

[0063] In step S410, a read of the target site may be obtained from the genome of the subject or pre-stored data. To obtain the above read, a variety of NGS methods may be used, but an amplicon NGS method is preferred.

[0064] In step S420, the obtained reads can be screened for reads having the same insert sequence based on a reference sequence. The reference sequence refers to a sequence of a known target site, and the reference sequence and the obtained reads can be aligned in a variety of ways, and the reads can be aligned according to the insert size.

[0065] Furthermore, in step S420, reads with soft-trimmed bases may be screened, where the meaning of soft-trimmed bases is as described above. To derive the soft-trimmed bases, reads may be aligned by bases.

[0066] In step S430, the part of the read that contains the soft-trimmed base sequence and / or the inserted sequence, or all of the parts, can be selected as SEED.

[0067] In step S440, the SEED obtained above can be used to analyze ITD, and the analysis can count the number of ITDs, and VAF can be derived by dividing the number of ITDs by the total number of ITDs. The clinical status of the patient can be predicted based on the VAF, for example, information related to the determination of the patient's disease can be provided, or the prognosis of a specific patient can be predicted, or information that can predict the patient's treatment responsiveness can be provided.

[0068] Figure 5 FIG. 4 is a flowchart for more specifically illustrating a SEED derivation method according to an embodiment.

[0069] Step S510 is a method for obtaining read information by an NGS method, and more specifically, the read information can be obtained by an amplicon NGS method.

[0070] Step S520 is a step for screening specific reads, and can screen reads having the same insertion sequence in more than three reads (step S520-1) and / or having the same soft-trimmed base sequence in more than three reads (step S520-2). The above steps can be performed independently or simultaneously.

[0071] Step S530 is a step for determining SEED. The sequence near the soft-trimmed base possessed by more than three reads containing the same soft-trimmed base sequence can be determined as SEED. More specifically, the bricklaying point of the soft-clipped segment, that is, the sequence adjacent to the 3' or 5' end of the soft-trimmed base can be determined as SEED. The above SEED may include the sequence adjacent to the 3' or 5' end. The above SEED includes part or all of the sequence of the soft-trimmed base, and the sequence length may be 12bp to 20bp.

[0072] Furthermore, the sequence near the insertion sequence possessed by three or more reads containing the same insertion sequence can be set as SEED, more specifically, the SEED contains all or part of the insertion sequence and the sequence adjacent to the 3' or 5' end of the insertion sequence, and the sequence length containing the sequence adjacent to the 3' or 5' end of the insertion sequence can be 12 bp to 20 bp. That is, it contains part or all of the insertion sequence and contains the sequence adjacent to the insertion sequence.

[0073] Figure 6 FIG. 6 is a block diagram of a SEED export device 600 according to an embodiment.

[0074] Reference Figure 6 , the device 600 may include a processor 610, a memory 620, and a display 630. In the above embodiment, the processor 610 may run with the device 600. However, the structural elements of the SEED derivation device 600 of one embodiment are not limited to the above example. According to other embodiments, the SEED derivation device 600 may include more structural elements or fewer structural elements than the above structural elements.

[0075] Processor 610 can obtain relevant information about the reads of any sequence through the NGS analysis method, screen the reads with the same insertion sequence in the above-obtained reads based on the reference sequence, and / or screen the reads with the same soft-trimmed bases, and select the part or all of the part of the above-screened reads containing the soft-trimmed base sequence and / or the insertion sequence as SEED.

[0076] In the step of screening the reads, when more than three reads have the same insertion sequence, the processor may screen the reads having the same insertion sequence, and when more than three reads have the same soft-trimmed base sequence, the processor may screen the reads.

[0077] The site containing the soft trimmed base sequence includes a sequence adjacent to the 3' or 5' end of the soft trimmed base, and the sequence length including the sequence adjacent to the 3' or 5' end of the soft trimmed base can be 12 bp to 20 bp.

[0078] The site containing the above-mentioned insertion sequence includes a sequence adjacent to the 3' or 5' end of the insertion sequence, and the sequence containing the sequence adjacent to the 3' or 5' end of the insertion sequence may have a length of 12 bp to 20 bp.

[0079] The memory 620 may store information about the reads, information about the reference sequence, and SEED.

[0080] Display 630 can display information about SEED or ITD, prognosis of disease, etc., such as Figure 5As described in, descriptive text about the SEED database (DB) can be provided.

[0081] The device of the present invention may include a processor, a memory for storing and executing program data, a permanent storage such as a disk drive, a communication port for communicating with an external device, a touch panel, a key, a button, and other user interface devices. The method implemented by the software module or algorithm can be stored in a computer-readable memory in the form of a computer-readable code or program instruction that can be run on the above-mentioned processor. Among them, the computer-readable memory includes magnetic storage media (for example, read-only memory (ROM), random-access memory (RAM), floppy disk, hard disk, etc.) and optical reading media (for example, CD-ROM, digital versatile disc (DVD)), etc. The computer-readable memory can be dispersed in a computer system connected by a network, and the medium can be read by a computer, stored in a memory, and run in a processor.

[0082] All documents cited in the present invention, including publications, patent applications, patents, etc., may be shown as if each reference is individually or specifically incorporated, or may be incorporated in the present invention in the same manner as if all of them were incorporated in the present invention.

[0083] To facilitate the understanding of the present invention, the preferred embodiments shown in the drawings are accompanied by figure marks, and specific terms are used to illustrate the embodiments of the present invention. However, the present invention is not limited by the specific terms. The present invention may include all structural elements that can be normally imagined by ordinary technicians in the technical field to which the present invention belongs.

[0084] The present invention can be shown by a functional frame structure or by a variety of processing steps. Such a functional frame structure can be implemented by a variety of hardware and / or software structures that perform specific functions. For example, the present invention can be composed of direct circuits such as memory, processing, logic, and look-up tables that can perform a variety of functions by the control of one or more microprocessors or by other control devices. The structural elements of the present invention are similar to those that can be performed by software programs or software elements. The present invention includes a variety of algorithms implemented by a combination of data structures, processing procedures, routines, or other program structures, which can be implemented by programming or scripting languages ​​such as C, C++, Java, and assemblers. The functional level can be implemented by algorithms running in one or more processors. In addition, the present invention can use existing technologies for electronic environment settings, signal processing, and / or data processing. Terms such as "mechanism", "element", "means", and "constitution" can be widely used and are not limited to mechanical and physical structures. The above terms can include the meaning of a series of processes (routines) of software connected to a processor, etc.

[0085] The specific operation described in the present invention is only an embodiment and is not intended to limit the present invention in any way. For the sake of brevity of the specification, the existing electronic structure, control system, software, and other functional aspects of the above system may be omitted. In addition, the line connections or connecting parts between the structural elements shown in the drawings are exemplary functional connections and / or physical or circuit connections, and can be replaced or shown with multiple functional connections, physical connections, or circuit connections in actual devices. In addition, if there is no specific description of "necessary", "important", etc., it may not be a structural element necessary for the application of the present invention.

[0086] In the specification of the present invention (especially the scope of the invention), the use of the term "above" and similar indicative terms may include the singular and the plural. Moreover, when the range is recorded in the present invention, it includes the invention of using a separate value belonging to the above range (if there is no record to the contrary), which is equivalent to recording the individual values ​​constituting the above range in the detailed description of the invention. Finally, if there is a clear order or no record to the contrary for the steps constituting the method of the present invention, the above steps can be performed in an appropriate order. The present invention is not necessarily limited to the order in which the above steps are recorded. The use of all examples or illustrative terms (for example, etc.) in the present invention is only used to explain the present invention in detail. As long as it is not limited to the scope of the invention, the scope of the present invention is not limited by the above examples or illustrative terms. Moreover, it is known to those skilled in the art of the present invention that various modifications, combinations and changes can be made according to design conditions and factors within the scope of the additional scope of the invention or its equivalent.

Claims

1. A method for deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method, characterized in that include: Step 1), obtaining reads by next generation sequencing methods; Step 2), a) screening the reads obtained above for reads having the same insertion sequence based on the reference sequence, and / or b) screening the reads having the same soft-trimmed bases; as well as Step 3), a part or all of the soft-trimmed base sequence and / or inserted sequence of the reads screened above is selected as SEED.

2. The method for deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method according to claim 1, characterized in that In the step of screening reads in step 2), if three or more reads have the same insert sequence, the reads having the same insert sequence are screened.

3. The method for deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method according to claim 1, characterized in that In the step of screening reads in the above step 2), if three or more reads have the same soft-trimmed base sequence, the reads having the same soft-trimmed base sequence are screened.

4. The method for deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method according to claim 1, characterized in that The portion containing the soft-trimmed base sequence in the above step 3) includes a sequence adjacent to the 3' or 5' end of the soft-trimmed base, and the sequence length of the above sequence adjacent to the 3' or 5' end of the soft-trimmed base is 12bp to 20bp.

5. The method of deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method according to claim 1, characterized in that The site containing the inserted sequence in the above step 3) contains a sequence adjacent to the 3' or 5' end of the inserted sequence, and the length of the sequence containing the sequence adjacent to the 3' or 5' end of the inserted sequence is 12 bp to 20 bp.

6. The method of deriving a sequence for analyzing internal tandem repeats in a next generation sequencing method according to claim 1, characterized in that The above-mentioned next-generation sequencing method is an amplicon-based next-generation sequencing method.

7. A method for analyzing internal tandem duplications in a next generation sequencing method, characterized in that include: Step 1), obtaining reads by next generation sequencing methods; Step 2), a) screening the reads obtained above for reads having the same insertion sequence based on the reference sequence, and / or b) screening the reads having the same soft-trimmed bases; Step 3), selecting a part or all of the soft-trimmed base sequence and / or inserted sequence of the reads screened above as SEED; as well as Step 4), query the above selected SEED, and for any read obtained by the next generation sequencing method, analyze the sequence matching the SEED.

8. The method for analyzing internal tandem duplications in a next generation sequencing method according to claim 7, characterized in that: The analysis in the above step 4) is a step of counting the number of matched sequences.

9. A device for deriving a sequence for analyzing internal tandem repeats in next generation sequencing analysis, characterized in that include: A processor, which obtains information about reads of an arbitrary sequence by a next generation sequencing method, screens the obtained reads for reads having the same insertion sequence based on a reference sequence, and / or screens the reads having the same soft-trimmed bases, and selects a part or all of the soft-trimmed base sequence and / or the insertion sequence of the screened reads as a SEED; A memory storing information related to the read, information related to the reference sequence and SEED; and A display showing information about the above exported SEED.

10. The device for deriving a sequence for analyzing internal tandem duplications in next generation sequencing analysis according to claim 9, characterized in that In the screening of the reads, if three or more reads have the same insert sequence, the reads having the same insert sequence are screened.

11. The device for deriving a sequence for analyzing internal tandem duplications in next generation sequencing analysis according to claim 9, characterized in that In the screening of the reads, if three or more reads have the same soft-trimmed base sequence, the reads having the same soft-trimmed base sequence are screened.

12. The device for deriving a sequence for analyzing internal tandem duplications in next generation sequencing analysis according to claim 9, characterized in that The site containing the soft-trimmed base sequence contains a sequence adjacent to the 3' or 5' end of the soft-trimmed base, and the sequence length of the sequence containing the sequence adjacent to the 3' or 5' end of the soft-trimmed base is 12 bp to 20 bp.

13. The device for deriving a sequence for analyzing internal tandem duplications in next generation sequencing analysis according to claim 9, characterized in that The site containing the above-mentioned insertion sequence includes a sequence adjacent to the 3' or 5' end of the insertion sequence, and the length of the sequence containing the sequence adjacent to the 3' or 5' end of the insertion sequence is 12 bp to 20 bp.

Images