KR20200106643A - High sensitive genetic variation detection and reporting system based on barcode sequence information - Google Patents

Abstract

The present invention relates to a highly sensitive genetic variation detection system based on barcode sequence information.
The present invention relates to a digital sequencing analyzer for detecting/analyzing genetic variation by performing digital sequencing on patient DNA obtained by a liquid biopsy method, and information on the genetic variation detected by the digital sequencing analyzer. It includes a service server that generates and provides a clinical support report related to the genetic mutation by referring to a public database.
According to the present invention, a liquid biopsy NGS data obtained by digital sequencing can be analyzed to detect genetic variation, and a report can be provided in a form that can be referred to by a medical professional for clinical decision making.

Description

High sensitivity genetic variation detection and reporting system based on barcode sequence information {HIGH SENSITIVE GENETIC VARIATION DETECTION AND REPORTING SYSTEM BASED ON BARCODE SEQUENCE INFORMATION}

The present invention relates to a highly sensitive genetic variation detection system based on barcode sequence information. More specifically, the present invention analyzes liquid biopsy NGS data obtained by digital sequencing to detect genetic mutations, and provides a report in a form that can be referred to by professional medical staff for clinical decision-making. It relates to a sensitive genetic variation detection system.

Important information related to diseases can be obtained through DNA sequence information of living organisms, and in recent years, the use of next generation sequencing (NGS) is increasing not only for mutation studies but also for clinical use.

With the introduction of the next-generation sequencing method, a number of genes related to the disease of interest are deviated from the conventional test method that directly probes several genes with a probe or uses a single marker to identify mutations depending on experiments. There has been a change in the trend of testing genes and various types of mutations.

In general, a method of collecting a sample to confirm mutations in a target gene related to cancer is a biopsy (biopsy) method in which DNA is obtained by removing tissue. Since this is a physical method, it causes pain in patients such as bleeding, inflammatory reactions and metastasis, and thus, a method of examining mutations by collecting DNA using a liquid biopsy, a non-invasive method, has become an issue.

In addition, the advantage of using a liquid biopsy is that it can reduce the risk and pain to the patient in a non-invasive method when periodic sampling is required for tolerance testing after drug prescription, and the disease may have already entered a certain stage. Research results have also been published indicating that an earlier examination is possible than image analysis and biopsy, which could be diagnosed at the time.

When tissue is collected by biopsy, it is possible to see the frequency of mutations in the local tissue, whereas liquid biopsy or cell-free DNA identifies mutations in the target gene to be identified among the total DNA mutations in the body. Since it must be done, the mutation frequency is inevitably low, and the low frequency mutation that exists at this time corresponds to the error rate (0.1%-1%) of the next-generation sequencing device.

In order to detect the actual variation outside the limit of the error rate, it is necessary to prove the existence of the variation by producing a quantity of data that can prove the existence of the actual variation, not the error, and to solve this, a deep dip that produces a large number of reads. Deep sequencing technology is required.

However, since deep sequencing increases production cost, it is not only burdensome for diagnostic testing, but also it is difficult to determine whether the detected mutation is an error generated during PCR (Polymerase Chain Reaction) or sequencing, or whether a mutation actually exists. This can lead to inaccurate results.

The digital sequencing method can avoid sequencing errors by attaching a unique random sequence (about 12 bp) called a UMI (Unique molecular index) to each read, amplifying PCR, and counting only UMI during quantitative analysis. have.

Therefore, a specialized solution to analyze liquid biopsy NGS data acquired by digital sequencing is required, which starts with registering patient information, uploads NGS data, evaluates data quality (Quality Control), and detects genetic variations. In addition, it must have a function to provide a report in a form that can be referred to for clinical decision-making by professional medical staff, but a solution with this function has not been provided until now.

Republic of Korea Patent Publication No. 10-2016-0020400 (published date: February 23, 2016, name: prediction method of single gene genetic variation in fetus using maternal serum DNA) Republic of Korea Patent Publication No. 10-2014-0023847 (published date: February 27, 2014, name Fetal genetic abnormality non-invasive detection)

The technical problem of the present invention is to analyze liquid biopsy NGS data acquired by digital sequencing to detect genetic variation, and to provide high sensitivity based on barcode sequence information that can be provided by reporting in a form that can be referenced by professional medical personnel for clinical decision making. It is to provide a genetic variation detection system.

In addition, the technical problem of the present invention is a cluster of reads containing barcode sequence information as a solution to the analysis algorithm when digital next-generation nucleotide sequence (Digital NGS) data using barcode sequences is produced using an analysis panel for liquid biopsy. ) To suppress the detection limit and NGS noise.

In addition, the technical problem of the present invention is to design an analysis pipeline that considers reads containing barcode sequence information, unlike read analysis through the existing next-generation sequencing method, thereby maintaining the advantages of liquid biopsy and disadvantages of next-generation sequencing methods. It complements the sensitivity of the identification of phosphorus mutations.

In addition, the technical problem of the present invention is to deviate from the conventional biopsy-based analysis method and to the patient's position, and through a system package equipped with a new analysis technique of a more convenient and safe non-invasive-next-generation sequencing method, cancer diagnosis or genetic marker It is to provide customized diagnostic solutions.

In addition, the technical problem of the present invention is to enable the analysis of both single-end and paired-end read data generated on different NGS sequencers, Ion torrent and Illumina-based platforms, in order to increase portability. Even if you are not familiar with NGS' professional analysis method, you can easily register samples and patients, conduct analysis, and return reports with a graphical user interface (GUI) on the web.

In addition, the technical task of the present invention is to generate a genetic mutation report including annotation information of mutations that can be applied to the clinic, so that a specialized medical center can be used as a basis for drugs or therapy for diseases related to mutations. To be.

The highly sensitive genetic mutation detection system based on barcode sequence information according to the present invention for solving these technical problems is digital sequencing that detects/analyzes genetic mutations by performing digital sequencing on patient DNA obtained by a liquid biopsy method. And a service server that generates and provides a clinical support report related to the genetic variation by referring to a public database for information on the genetic variation detected by the analysis analyzer and the digital sequencing analyzer.

In the highly sensitive genetic variation detection system based on barcode sequence information according to the present invention, the service server includes a patient information management unit that receives and manages patient information, and NGS data, which is a result of Next Generation Sequencing (NGS) analysis of the patient DNA. Upload and evaluate the quality of the NGS data (Quality Control), if the foam quality of the NGS data meets the acceptance criteria, transfer the NGS data to the digital sequencing analyzer to detect/analyze the genetic variation of the patient DNA The NGS data management unit for requesting a request, receives information on the genetic variation of the patient's DNA from the digital sequencing analyzer, and collects detailed description information about the genetic variation by referring to the variation information DB published on the Internet. It characterized in that it comprises a report management unit that generates and provides a clinical support report related to the genetic variation by processing the detailed description information on the genetic variation collected by the unit and the information collection unit in a predefined format.

In the highly sensitive genetic variation detection system based on barcode sequence information according to the present invention, the clinical support report includes information on a patient who has provided DNA, information specifying a DNA sample, and a reference standard in which the genetic variation is defined in HG19 or HG38. It is characterized by including information on the frequency of mutations compared to the sequence (the ratio of all unique barcodes including mutations, variant MT (barcode) fraction, VMF).

In the high-sensitivity genetic variation detection system based on barcode sequence information according to the present invention, the report management unit determines the relationship between the genetic variation and the phenotype through the clinical support report according to the guideline presented in the variation information DB. It is characterized in that it is divided into a plurality of taxa and provided based on information on pathogenicity, activity, and actionability.

In the highly sensitive genetic variation detection system based on barcode sequence information according to the present invention, the digital sequencing analyzer includes a read generation process for generating a read including a barcode by performing digital sequencing on the patient DNA, A trimming process for removing an adapter sequence and a low quality lead from the read, and a read from which the adapter sequence and a low quality lead are removed are representative of the target species. A process of mapping a reference sequence attached to a reference sequence, which is a sequence, searching and defining a barcode sequence based on the mapped information of the reads, and clustering the reads based on the defined barcode sequence. It characterized by performing a clustering process and a genetic variation detection process of detecting a genetic variation in reads having the same barcode among reads clustered based on the barcode sequence.

According to the present invention, high sensitivity genetics based on barcode sequence information that can be provided by analyzing liquid biopsy NGS data acquired by digital sequencing to detect genetic mutations, and to provide a report in a form that can be referenced by professional medical staff for clinical decision making. There is an effect that a mutation detection system is provided.

In addition, when digital next-generation nucleotide sequence (Digital NGS) data using barcode sequence is produced using an analysis panel for liquid biopsy, a cluster between reads containing barcode sequence information is created as a solution to the analysis algorithm to limit detection and NGS noise can be suppressed.

In addition, unlike read analysis through the existing next-generation sequencing method, by designing an analysis pipeline that considers reads containing barcode sequence information, it is possible to supplement the sensitivity of mutation identification, which is a disadvantage, while maintaining the advantages of liquid biopsy.

In addition, through a system package equipped with a new analysis technique of a more convenient and safe non-invasive-next-generation sequencing method, it will provide a customized diagnosis solution using a genetic marker or a cancer diagnosis from the patient's point of view beyond the conventional biopsy method. I can.

In addition, in order to increase the portability, we designed a system that supports analysis of both single-end and paired-end read data generated on different NGS sequencers, Ion torrent and Illumina-based platforms. , Even if you are not familiar with NGS' method of performing professional analysis, you can support to easily perform registration, analysis, and report return of samples and patients with a graphical user interface (GUI) on the web.

In addition, by generating a genetic mutation report including annotation information of mutations that can be applied to the clinic, it is possible to support medical clinics specializing in mutation-related diseases to be used as basis data for drugs or therapy.

1 is a diagram showing a high sensitivity genetic variation detection system based on barcode sequence information according to an embodiment of the present invention,
2 is a diagram illustrating an exemplary operation of a system for detecting high sensitivity genetic variation based on barcode sequence information according to an embodiment of the present invention.
3 is a diagram for conceptually explaining error detection in a read using barcode sequence information in an embodiment of the present invention,
4 is a diagram illustrating an example of a sample registration screen according to an embodiment of the present invention,
5 is a diagram illustrating an example data upload screen in an embodiment of the present invention,
6 is a diagram illustrating an exemplary received sample and analysis status screen in an embodiment of the present invention,
7 is a diagram illustrating an analysis information and a report download screen according to an embodiment of the present invention,
8 is a diagram illustrating a screen of a clinical support report that is a result of an analysis of genetic variation according to an embodiment of the present invention.

Specific structural or functional descriptions of the embodiments according to the concept of the present invention disclosed in the present specification are only exemplified for the purpose of describing the embodiments according to the concept of the present invention, and the embodiments according to the concept of the present invention are in various forms. And are not limited to the embodiments described herein.

Since the embodiments according to the concept of the present invention can apply various changes and have various forms, embodiments are illustrated in the drawings and will be described in detail in the present specification. However, this is not intended to limit the embodiments according to the concept of the present invention to specific disclosed forms, and includes all changes, equivalents, or substitutes included in the spirit and scope of the present invention.

Terms such as first or second may be used to describe various elements, but the elements should not be limited by the terms. The terms are only for the purpose of distinguishing one component from other components, for example, without departing from the scope of the rights according to the concept of the present invention, the first component may be named as the second component and similarly the second component. The component may also be referred to as a first component.

When a component is referred to as being "connected" or "connected" to another component, it should be understood that it is directly connected or may be connected to the other component, but other components may exist in the middle. will be. On the other hand, when a component is referred to as being "directly connected" or "directly connected" to another component, it should be understood that there is no other component in the middle. Other expressions describing the relationship between components, such as "between" and "directly between" or "adjacent to" and "directly adjacent to" should be interpreted as well.

Terms used in the present specification are used only to describe specific embodiments, and are not intended to limit the present invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In the present specification, terms such as "comprise" or "have" are intended to designate the presence of features, numbers, steps, actions, components, parts, or combinations thereof described herein, but one or more other features. It is to be understood that the possibility of addition or presence of elements or numbers, steps, actions, components, parts, or combinations thereof is not preliminarily excluded.

Unless otherwise defined, all terms, including technical or scientific terms, used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. Terms as defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related technology, and should not be interpreted as an ideal or excessively formal meaning unless explicitly defined in the present specification. .

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a diagram showing a high sensitivity genetic variation detection system based on barcode sequence information according to an embodiment of the present invention, and Figure 2 is an exemplary view of a high sensitivity genetic variation detection system based on barcode sequence information according to an embodiment of the present invention. 3 is a diagram for conceptually explaining detection of errors in a read using barcode sequence information in an embodiment of the present invention, and FIG. 4 is a diagram illustrating a sample in an embodiment of the present invention. FIG. 5 is a diagram illustrating a registration screen as an example, FIG. 5 is a diagram illustrating a sample data upload screen in an embodiment of the present invention, and FIG. 6 is a diagram illustrating a received sample and An exemplary view showing an analysis status screen, FIG. 7 is a diagram illustrating an analysis information and a report download screen according to an embodiment of the present invention, and FIG. 8 is a diagram illustrating an oil field in an embodiment of the present invention. This is an exemplary diagram showing the clinical support report screen, which is the result of mutation analysis.

Referring to FIGS. 1 to 8, a high sensitivity genetic variation detection system based on barcode sequence information according to an embodiment of the present invention includes a digital sequencing analyzer 20 and a service server 30.

The digital sequencing analyzer 20 performs a function of detecting/analyzing a genetic variation by performing digital sequencing on patient DNA obtained by a liquid biopsy method.

For example, the digital sequencing analyzer 20 performs digital sequencing on patient DNA to generate a read including a barcode, and an adapter sequence and low quality from the read. quality) A trimming process for removing reads, and reference sequence mapping in which the adapter sequences and reads from which low quality reads are removed are attached to a reference sequence, which is a representative sequence of the target species ( mapping) process, a clustering process of searching and defining a barcode sequence based on the mapped information of the reads, clustering the reads based on the defined barcode sequence, and among the reads clustered based on the barcode sequence, It can be configured to perform a genetic variation detection process that detects genetic variation in reads with the same barcode.

The service server 30 performs a function of generating and providing a clinical support report related to the genetic variation by referring to a public database for information on the genetic variation detected by the digital sequencing analyzer 20.

For example, the service server 30 may include a patient information management unit 310, an NGS data management unit 320, an information collection unit 330, and a report management unit 340.

The patient information management unit 310 performs a function of receiving and managing patient information.

The NGS data management unit 320 uploads NGS data, which is a result of Next Generation Sequencing (NGS) analysis of patient DNA, and evaluates the quality of the NGS data, so that if the NGS data meets the acceptance criteria, the digital Transmits NGS data to the sequencing analyzer 20 to perform a function of requesting detection/analysis of genetic mutations on patient DNA.

The information collection unit 330 receives information on the genetic variation of patient DNA from the digital sequencing analyzer 20, and collects detailed explanatory information on the genetic variation by referring to the variation information DB 40 published on the Internet. Performs the function of

The report management unit 340 performs a function of generating and providing a clinical support report related to the genetic variation by processing detailed description information on the genetic variation collected by the information collection unit 330 in a predefined format.

For example, in the clinical support report generated and provided by the report management unit 340, information on a patient who provided DNA (name, date of birth, etc.), information specifying a DNA sample (sample ID, type classification, registration date, Date of analysis, etc.), and information on the mutation frequency (ratio of all unique barcodes containing mutations, Variant MT (barcode) fraction, VMF) compared to the reference sequence defined in HG19 or HG38. have.

For example, the report management unit 340, through a clinical support report, determines the relationship between genetic variation and phenotype in accordance with the guidelines presented in the variation information DB 40, in accordance with the clinical significance (pathogenicity), activity, and risk (actionability). ) May be configured to be divided into a plurality of taxa and provided based on the information. As a specific example, in order to interpret the clinical implications of genetic mutations, standard guidelines suggested by specific institutions based on clinical pathogenicity, activity, and actionability (for example, ACMG guideline, https: //www.ncbi.nlm.nih.gov/pmc/articles/PMC4544753/), it can be divided into five taxa as follows.

1.Pathogenic-strong clinical significance and activity

2.Likely pathogenic-potential clinical significance and activity

3. Likely benign-a mutation that is likely to be benign

4.Benign-a positive mutation

5.Uncertain significance-A variation that does not have enough evidence to classify it into the preceding four categories.

The user terminal 10 is a means for accessing the service server 30 and may be, for example, a terminal used by a doctor or the like.

Hereinafter, a detailed configuration of a high-sensitivity genetic variation detection system based on barcode sequence information according to an embodiment of the present invention will be exemplarily described.

1. Composition and features

The highly sensitive genetic variation detection system based on barcode sequence information according to an embodiment of the present invention pre-processes a read including barcode sequence information by using adapter sequence information including barcode sequence information in addition to the existing next-generation sequencing method, and Detect mutations based on sequence information. By using this, since amplification errors and sequencing errors that may occur from the library to the sequence determination step can be identified, it is possible to overcome the problem of detection limit, a disadvantage of liquid biopsy. The adapter sequence is a short known DNA sequence that is attached to both ends of the target DNA. The primer sequence is complementarily bound to any part of the adapter sequence and is sequenced.

The highly sensitive genetic mutation detection system based on barcode sequence information according to an embodiment of the present invention determines the authenticity of the mutation, calculates the allele frequency of the mutation, and interlocks with the DB containing the detected mutation information. Registers patient information and puts DNA in the sequencer, as well as the components of the pipeline for analyzing specialized genomic bioinformation, and when the analysis is complete, the initial data generated by the sequencer is uploaded to the cloud analysis environment and a series of biological information analysis of the pipeline Finally, it includes a management and reporting system component, that is, a service server 30, that generates a clinically applicable mutation report in PDF format.

2. Configuration of highly sensitive genetic mutation detection system based on barcode sequence information

The highly sensitive genetic variation detection system based on barcode sequence information according to an embodiment of the present invention has two major components, namely, a digital sequencing analyzer 20 that performs genetic variation detection and analysis, and sample registration, analysis processing, and reporting management. It can be divided into a service server 30 to perform.

end. Genetic variation detection analysis process

The genetic variation detection and analysis process of the highly sensitive genetic variation detection system based on barcode sequence information may be performed according to E in the flow illustrated in FIG. 2. The analysis method may include a barcode sequence information clustering step, which is distinct from the analysis process of general Whole Genome Sequencing (WGS) or Whole Exome Sequencing (WES) in order to improve the sensitivity of liquid biopsy.

[Overall Course Summary]

In step (1), digital sequencing is performed to generate a read including barcode sequence information (S160).

Specifically, in process (1), DNA extracted from a liquid biopsy is separated from only the target genes from the process of DNA library preparation (library prep), attaching the necessary sequences necessary for the next-generation sequencing method, and performing various amplification processes. It goes through the process of creating target genes. Thereafter, when the target gene is inserted into the sequencer, sequences that are complementary to the target genes are attached to determine the nucleotide sequence of short strands.The short sequences thus generated are called reads, and the sequencer stores each nucleotide sequence of the generated reads. have.

In step (2), a high quality lead is selected through a process of removing an adapter sequence and a low quality lead (S170).

Specifically, in the process (2), each of the corresponding reads has quality information, and since the quality of the reads that are not clearly read is degraded, these leads, which may cause sequencing errors, are removed in advance. In addition, since only the sequence information of the target gene is required during this process, the adapter sequence attached to the library is also removed.

Process (3). The reads including the barcode sequence information go through a mapping process that attaches them to a reference sequence that can be called a representative sequence of the target species (S180).

Specifically, in step (3), a mapping process is performed in which reads having only the sequence of the target gene are attached to the reference sequence. Mapping can be performed using the BWA-mem program, which is a sequence alignment tool that is fast, accurate, and has a relatively low error rate. Generally, after this process, multiple reads are attached to the desired target sequence. You will be able to confirm. However, in the process, if the read sequence has many repetitive sequences or similar sequences exist in other parts of the reference sequence, it may be mapped to the wrong part. In this case, incorrect mutation information is obtained, and of course, the error rate in variant calling is reduced. It is raised.

As illustrated in FIG. 3, reads produced by digital sequencing contain a series of barcode sequence information sequences that are not related to the genetic information of the experimental sample, which includes a mutation region of interest before producing NGS data. This is the ID sequence given when creating a library for genomic DNA or target sequence. If there is a sequence of barcode sequence information, when amplification occurs during the NGS sequencing process or after mapping, the reads that have the same barcode sequence information sequence are investigated, and the incorrectly polymerized base is treated as a sequencing error, and the mapping is incorrect. Leads from from can also be removed.

In step (4), the barcode sequence information sequence is searched for and defined based on the mapped information of the read, and the read is clustered based on the defined barcode sequence information sequence (S190, S200, S210).

Specifically, when analyzing the mutation in step (4), it is possible to determine whether the detected mutation is due to PCR or sequencing errors or whether the mutation actually exists through comparison between reads having the same barcode sequence information. Afterwards, the process of extracting the barcode sequence information sequence from all reads and clustering the reads of the same barcode sequence information must be carried out, and the above process can be divided into four detailed steps.

(4-1) The location of the barcode sequence information in the read sequence is the FLAG column, POS column, and CIGAR of the bam format (https://samtools.github.io/hts-specs/SAMv1.pdf) file created through reference mapping. Predict through columns. The FLAG column describes whether the read is mapped and the orientation, and the POS column describes where the read is mapped to the reference sequence. And the CIGAR column means how the reads are mapped.

(4-2) Assuming that the barcode sequence information is located in the front of the read, the position where the mapping of the read started is identified through the POS and CIGAR columns. The position from the mapping start position to the forward direction up to 12bp (barcode sequence information consists of 12 random sequences) is predicted by barcode sequence information. At this time, it is determined whether the read is in the reverse direction through the FLAG column.

(4-3) After determining the position of the barcode sequence information from each read, the sequence of the barcode sequence information is extracted, and the reads are grouped based on the extracted barcode sequence information. This process is based on the method of clustering the papers (refer to the current status of the paper, Peng et al. BMC Genomics, 2015), but optimized the sequence processing method according to the characteristics of the data.

(4-4) After going through the clustering process, a new bam file is created by modifying the read ID of the existing bam file to a form containing barcode sequence information.

In step (5), a mutation is detected in a read having the same barcode sequence information by using a barcode sequence information-aware variant caller (S220).

Specifically, in step (5), an embodiment of the present invention uses a barcode-aware variant calling module based on barcode sequence information.

The general mutation analysis pipeline is not optimized for data analysis generated from digital sequencing, but an embodiment of the present invention is based on smCounter, a molecular barcode-aware variant caller that can perform mutation analysis using barcode sequence information. Thus, a mutation analysis pipeline was established.

Since smCounter operates only with paired-end leads, it has been supplemented to work with single-end leads.

Using the alignment information of the target area, reads within the area are retrieved, and reads with the same barcode sequence information are collected to calculate Barcode depth (MT depth), quality, pi (prediction index), etc., and finally discovered mutations Is output as a VCF format file.

In step (6), mutation description information is searched for the detected mutation in a public database.

Specifically, in step (6), mutation annotation information is attached by referring to the genetic mutation DB published on the Internet.

The primary result file is generated by extracting only information that can be used clinically from a genetic variation DB (e.g., ClinVar, OMIM, dbSNP, mycancergenome, etc.) containing useful mutation information for the identified mutations in the sample.

The data that can be used include ClinVar's variant classification, NM ID, gDNA ID and dbSNP's rs number, mycancergenome's theraphy information, COSMIC's prediction, and drug resistance, but are not limited thereto.

I. Sample registration, analysis processing and reporting management

As illustrated in FIGS. 4 to 8, the service server 30 is responsible for a function of outputting the structure and management functions and results of all systems except for genetic variation analysis in a report format. It consists of a total of 4 functions.

For example, referring to FIG. 4, the service server 30 may support a patient and sample registration function.

Patient information can be entered individually or registered in a batch as a file conforming to the specified Excel format.

For example, referring to FIG. 5, the service server 30 may support an automatic delivery function of next-generation sequence data generated by a cloud analysis server.

Since the NGS test result file is large (0.3 ~ 2 GB per sample), a separate large file transfer function is installed. This function is divided into “Local” function for when the device is in the local network, and “GridFTP, S3” function for when the device is not. By registering a specific directory, the file transfer operation is automatically started.

For example, referring to FIGS. 6 and 7, the service server 30 supports a mutation analysis progress check and analysis case management function.

When the upload of the liquid biopsy NGS data of the registered patient is completed, the sample status information is changed to a state in which QC analysis can be performed. After the QC analysis, if it is confirmed that it is NGS data of an appropriate quality, it is changed to a state in which digital sequencing mutation analysis can be performed, and analysis can be performed. When the analysis is complete, you can download the result report from the detailed information.

For example, referring to FIG. 8, the service server 30 provides a report that can be used clinically, that is, a clinical support report output function.

For example, a clinical support report may include patient information (name, date of birth, etc.) and DNA sample information (sample ID, type classification, registration date, analysis date, etc.) who provided DNA.

Also, for example, the identified mutation is a wild type within the sample, e.g., the frequency of mutation (the ratio of the mutation in the entire unique barcode, compared to the reference sequence defined in HG19 or HG38, Variant MT (barcode) fraction, VMF) can be checked.

In addition, for example, annotative information in the genetic variation DB is used to indicate the variant classification of each genetic variation DB, and by using this information, a specialized medical center can diagnose the test result to the patient. The mutation of interest for each gene can be identified, and it can be configured to help with the prescription based on other annotation information and prescription therapies of the mutation.

As described in detail above, according to the present invention, a barcode that can be provided by analyzing liquid biopsy NGS data acquired by digital sequencing to detect genetic mutations, and to provide a report in a form that can be referred to by a medical professional for clinical decision making. There is an effect of providing a high-sensitivity genetic mutation detection system based on sequence information.

In addition, when digital next-generation nucleotide sequence (Digital NGS) data using barcode sequences is produced using an analysis panel for liquid biopsy, a cluster between reads containing barcode sequence information is created as a solution to the analysis algorithm to limit detection and It suppresses NGS noise.

In addition, unlike read analysis through the existing next-generation sequencing method, by designing an analysis pipeline that considers reads containing barcode sequence information, it is possible to supplement the sensitivity of mutation identification, which is a disadvantage, while maintaining the advantages of liquid biopsy.

In addition, through a system package equipped with a new analysis technique of a more convenient and safe non-invasive-next-generation sequencing method, it will provide a customized diagnosis solution using a genetic marker or a cancer diagnosis from the patient's point of view beyond the conventional biopsy method. I can.

In addition, in order to increase the portability, we designed a system that supports analysis of both single-end and paired-end read data generated on platforms based on different NGS sequencers, Ion torrent and Illumina. , Even if you are not familiar with NGS' method of performing professional analysis, you can support to easily perform registration, analysis, and report return of samples and patients with a graphical user interface (GUI) on the web.

In addition, by generating a genetic mutation report including annotation information of mutations that can be applied to the clinic, it is possible to support medical clinics specializing in mutation-related diseases to be used as evidence for drugs or therapy.

10: user terminal
20: Analyzer for digital sequencing
30: service server
40: mutation information DB
310: Patient Information Management Department
320: NGS data management unit
330: information collection unit
340: report management unit

Claims (5)

As a highly sensitive genetic variation detection system based on barcode sequence information,
A digital sequencing analyzer that detects/analyzes genetic variation by performing digital sequencing on patient DNA obtained by a liquid biopsy method; And
High sensitivity genetic variation detection based on barcode sequence information, including a service server that generates and provides a clinical support report related to the genetic variation by referring to a public database for information on the genetic variation detected by the digital sequencing analyzer system. The method of claim 1,
The service server,
A patient information management unit receiving and managing patient information;
When the NGS data, which is the result of the NGS (Next Generation Sequencing) analysis of the patient DNA, is uploaded, and the quality of the NGS data is evaluated (Quality Control), and the foam quality of the NGS data meets the acceptance criteria, the digital sequencing analyzer An NGS data management unit for requesting detection/analysis of the genetic variation of the patient DNA by transmitting the NGS data to the patient;
An information collection unit receiving information on the genetic variation of the patient's DNA from the digital sequencing analyzer and collecting detailed description information about the genetic variation by referring to the variation information DB published on the Internet; And
It characterized in that it comprises a report management unit that generates and provides a clinical support report related to the genetic variation by processing the detailed description information on the genetic variation collected by the information collection unit in a predefined format, barcode sequence information Based high sensitivity genetic variation detection system. The method of claim 2,
In the above clinical support report,
Information on the patient who provided the DNA, information specifying the DNA sample, and the frequency of mutation (the ratio of the mutation in the entire unique barcode) compared to the reference reference sequence defined in HG19 or HG38, Variant MT (barcode ) Fraction, VMF), characterized in that it contains information, barcode sequence information based high sensitivity genetic variation detection system. The method of claim 3,
The report management unit,
Through the clinical support report, the relationship between the genetic mutation and the phenotype is divided into a plurality of taxa based on information on clinical significance, activity, and actionability according to the guidelines presented in the mutation information DB. Characterized in that provided by, high-sensitivity genetic variation detection system based on barcode sequence information. The method of claim 1,
The digital sequencing analyzer,
A read generation process of generating a read including a barcode by performing digital sequencing on the patient DNA; and
A trimming process of removing an adapter sequence and a low quality lead from the lead, and
A reference sequence mapping process in which the adapter sequence and the reads from which the low quality reads are removed are attached to a reference sequence, which is a representative sequence of a target species,
A clustering process of searching and defining a barcode sequence based on the mapped information of the reads, and clustering the reads based on the defined barcode sequence,
High sensitivity genetic variation detection system based on barcode sequence information, characterized in that performing a genetic variation detection process for detecting genetic variation in reads having the same barcode among reads clustered based on the barcode sequence.

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