WO2014005329A1

WO2014005329A1 - Method and system for determining integration manner of foreign gene in human genome

Info

Publication number: WO2014005329A1
Application number: PCT/CN2012/078311
Authority: WO
Inventors: 曾玺; 李伟阳; 陈盛培; 蒋慧; 汪建; 王俊; 杨焕明; 张秀清
Original assignee: 深圳华大基因科技有限公司; 深圳华大基因研究院
Priority date: 2012-07-06
Filing date: 2012-07-06
Publication date: 2014-01-09
Also published as: CN104428423A

Abstract

Provided are a method, system, and readable medium for determining an integration manner of a foreign gene in a human genome. The method for determining an integration manner of a foreign gene in a human genome comprises: capturing, by using a capture probe, an integrated DNA fragment possibly containing a foreign gene fragment from a human genome nucleic acid sample; performing sequencing for the captured DNA fragment to obtain a sequencing result; performing first purification on the sequencing result; performing first comparison on the sequencing result obtained through the first purification and a known human genome sequence and a foreign gene sequence to obtain sequencing data possibly containing a foreign gene integration fragment; assembling the sequencing data possibly containing a foreign gene integration fragment to obtain an assembling result; performing second purification on the assembling result; and performing second comparison on the assembling result obtained through the second purification, and determining an integration manner of a foreign gene in a human genome based on a second comparison result.

Description

Method and system for determining the manner in which foreign genes are integrated in the human genome

Priority information

No technical field

The present invention relates to the field of biotechnology, and in particular, to a bioinformatics analysis method for detecting the integration of a pathogen genome in a human genome, and more particularly, to a method for determining a method for integrating a foreign gene in a human genome , system and computer readable media. Background technique

It is known that HBV, HIV, and HPV viruses can integrate their own DNA into the human genome. After HBV infection, the virus causes inflammation of the infected site through replication, which in turn triggers cell cancer. The high-risk type HPV16 in the HPV virus causes inflammation in the HPV patients after cervical infection, and promotes the abnormal growth of cervical cells, resulting in canceration. The integrated imaging of HPV16 is evident in the cancerous tissue.

Over the years, the method of genetic integration has been studied, and PCR has been used to detect the integration status of HPV16 virus in cervical cancer and precancerous lesions. Among them, alu and other methods have been widely used. With the development of high-throughput sequencing technology, the improvement of high-throughput sequencing and information analysis methods provides a basis for analyzing the integration of pathogens. The commonly used bioinformatics analysis is mainly limited. The pair end sequence is aligned, and the approximate insertion position is determined by the alignment position of the PE reads, and the exact position cannot be determined. Therefore, the current research methods are still to be improved. Summary of the invention

The present invention aims to solve at least one of the technical problems existing in the prior art. The present invention aims to propose a method for efficiently identifying integration sites of precise pathogen genomic fragments within the genome-wide range.

In one aspect of the invention, the invention proposes a method of determining the manner in which a foreign gene is integrated in the human genome. According to an embodiment of the present invention, the method comprises: capturing a DNA fragment that may contain an integration of a foreign gene fragment from a human genomic nucleic acid sample using a capture probe; sequencing the captured DNA fragment to obtain a plurality of sequencing data a sequencing result; performing a first impurity removal on the sequencing result to obtain a sequencing result of the first impurity removal; performing the first impurity-clearing sequencing result with a known human genome sequence and a foreign gene sequence First alignment to obtain sequencing data that may contain an integrated fragment of a foreign gene; assembling the obtained sequencing data that may contain an integrated fragment of the foreign gene to obtain an assembly result, the assembly result being composed of a plurality of assembly data Performing a second impurity removal on the assembly result to obtain a second impurity-free assembly result; performing a second ratio of the second impurity-free assembly result to a known human genome sequence and a foreign gene sequence And, based on the second alignment result, determining the foreign gene in the human genome Integrated way. This method can effectively determine the way in which foreign genes such as pathogen genomes are integrated in the human genome.

In a second aspect of the invention, the invention also provides a method for determining the integration of a foreign gene in the human genome System. According to an embodiment of the invention, the system comprises: a capture device adapted to capture, from a human genomic nucleic acid sample, a DNA fragment that may contain an integration of a foreign gene fragment using a capture probe; a sequencing device, the sequencing device The capture device is coupled and adapted to sequence the captured DNA fragments to obtain sequencing results consisting of a plurality of sequencing data; a first impurity removing device, the first impurity removing device being coupled to the sequencing device, And adapted to perform the first impurity removal on the sequencing result, so as to obtain the sequencing result after the first impurity removal; the first comparison device, the first comparison device is connected to the first impurity removal device, and is suitable Performing a first alignment of the first impurity-cleared sequencing result with a known human genome sequence and a foreign gene sequence to obtain sequencing data that may contain an integrated fragment of the foreign gene; assembling device, the assembling device Connected to the first alignment device and adapted to assemble the resulting sequencing data that may contain foreign gene integration fragments In order to obtain an assembly result, the assembly result is composed of a plurality of assembly data; a second impurity removal device, the second impurity removal device is connected to the assembly device, and is adapted to perform second impurity removal on the assembly result, In order to obtain the assembly result after the second impurity removal; the second comparison device, the second comparison device is connected to the second impurity removal device, and is adapted to combine the result of the second impurity removal assembly with A second alignment of the known human genome sequence and the foreign gene sequence; and an analysis device adapted to determine the manner in which the foreign gene is integrated in the human genome based on the second alignment result. With the system according to an embodiment of the present invention, the aforementioned method can be effectively implemented, whereby the manner in which a foreign gene such as a pathogen genome is integrated in the human genome can be efficiently determined.

In yet another aspect of the invention, the invention provides a computer readable medium. According to an embodiment of the invention, instructions are stored on the computer readable medium, the instructions being adapted to be executed by the processor to determine how the foreign gene is integrated in the human genome by the following steps: performing a first impurity removal on the sequencing result In order to obtain the sequencing result of the first impurity removal; the first alignment result of the first impurity is first aligned with the known human genome sequence and the foreign gene sequence, so as to obtain an integration fragment which may contain the foreign gene Sequencing data; assembling the obtained sequencing data, which may contain the integrated fragment of the foreign gene, to obtain an assembly result, the assembly result being composed of a plurality of assembly data; performing second impurity removal on the assembly result to obtain Performing a second impurity-free assembly result; and performing a second alignment of the second impurity-free assembly result with a known human genome sequence and a foreign gene sequence, and determining based on the second alignment result The manner in which the foreign gene is integrated in the human genome, wherein the sequencing result is obtained by the following Obtained: A capture probe is used to capture a DNA fragment that may contain an integration of a foreign gene fragment from a human genomic nucleic acid sample; the captured DNA fragment is sequenced to obtain a sequencing result composed of a plurality of sequencing data. The computer readable medium can be used to efficiently determine the integration of a foreign gene, such as a pathogen genome, in the human genome.

The additional aspects and advantages of the invention will be set forth in part in the description which follows. DRAWINGS

The above and/or additional aspects and advantages of the present invention will become apparent and readily understood from

1 is a flow chart of a method for determining a manner of integration of a foreign gene in a human genome according to an embodiment of the present invention. Schematic diagram

2 is a flow chart showing a method of determining a manner of integration of a foreign gene in a human genome according to still another embodiment of the present invention;

Figure 3 is a schematic illustration of the structure of a system for determining the manner in which foreign genes are integrated in the human genome, in accordance with one embodiment of the present invention. detailed description

The embodiments of the present invention are described in detail below, and the examples of the embodiments are illustrated in the drawings, wherein the same or similar reference numerals are used to refer to the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the drawings are intended to be illustrative of the invention and are not to be construed as limiting.

The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implied the number of technical features indicated. Thus, features defining "first" and "second" may include one or more of the features, either explicitly or implicitly. In the description of the present invention, the meaning of "plurality" is two or more, unless specifically defined otherwise. As used herein, the terms "above" and "below" include the number, for example "80% or more" means >80%, and "2% or less" means <2%. PER Assembly: As described herein, PER refers to the assembly of pair end sequence data. That is, each pair of PE sequencing data obtained by pair end sequencing is assembled according to the overlapping relationship between the sequences. Substitution: As described herein, a segment of a genomic DNA fragment of a pathogen is inserted into the human genome, and the deletion of the human genomic DNA at the insertion site is called a substitution. PCR repeat: repeated amplification during PCR. Adaptor: Sequencing connector, adaptor will appear in the sequence data of some of the lower machines. BWA: Short for Burrows-Wheeler Aligner, a sequence alignment software. Soap: short for Short Oligonucleotide Analysis Package, is a comparison software.

In the present invention, the terms "connected", "connected" and the like should be understood broadly, and may be, for example, a fixed connection, a detachable connection, or an integral connection, unless otherwise explicitly stated and defined; The mechanical connection may also be an electrical connection; it may be directly connected, or may be indirectly connected through an intermediate medium, and may be internal communication between the two elements. For those of ordinary skill in the art, the specific meaning of the above terms in the present invention can be understood on a case-by-case basis. Method for determining the method of exogenous gene in the human genome

According to a first aspect of the invention, the invention proposes a method of determining the manner in which a foreign gene is integrated in the human genome. According to an embodiment of the present invention, referring to FIG. 1, the method includes:

Capture step S100: Capture a DNA fragment that may contain an integration of a foreign gene fragment from a human genomic nucleic acid sample using a capture probe. According to an embodiment of the present invention, the type of the foreign gene that can be analyzed using the method of the present invention is not particularly limited. As long as it can be integrated with the human genome, and its gene sequence can be obtained or already known. According to an embodiment of the invention, the foreign gene that can be studied is the pathogen genome. Further, according to a specific example of the present invention, the pathogen is HBV. Thereby, the integration of pathogens such as HBV with the human genome can be effectively analyzed. Sequencing step S200: Sequencing the captured DNA fragments to obtain sequencing results consisting of multiple sequencing data. According to an embodiment of the present invention, the manner of sequencing the captured DNA fragments is not particularly limited. According to an embodiment of the invention, sequencing is performed by a second generation sequencing platform. According to an embodiment of the invention, the whole genome sequencing library can be sequenced using at least one selected from the group consisting of Hiseq2000, SOLiD, 454 and single molecule sequencing devices. Thereby, the efficiency of determining the integration mode of the foreign gene in the human genome can be further improved by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices. Of course, those skilled in the art will appreciate that other sequencing methods and devices can be used for whole genome sequencing, such as third generation sequencing techniques, as well as more advanced sequencing techniques that may be developed in the future. According to an embodiment of the present invention, the length of the sequencing data obtained by whole genome sequencing is not particularly limited. According to an embodiment of the present invention, it is preferable that the sequencing length is lOObp, whereby the analysis effect can be further improved.

The first impurity removing step S300: performing first impurity removal on the sequencing result to obtain a sequencing result after the first impurity removal. According to an embodiment of the present invention, the type of the first impurity removal is not particularly limited. For example, the first impurity removal may further include removing PCR duplication, removing low quality sequencing data, and removing at least one of the linker-containing sequencing data. Kind. Thereby, the analysis efficiency can be further improved.

The first alignment step S400: the first impurity-sequencing sequencing result is first aligned with the known human genome sequence and the foreign gene sequence to obtain sequencing data which may contain the foreign gene integration fragment. According to an embodiment of the invention, the first alignment can be performed using SOAP. Thereby, the analysis efficiency can be further improved.

Assembly step S500: The obtained sequencing data, which may contain the foreign gene integration fragment, is assembled to obtain an assembly result, which is composed of a plurality of assembly data. According to an embodiment of the invention, the assembly is performed by based on an overlapping relationship between the sequenced data.

The second impurity removing step S600: performing second impurity removal on the assembly result to obtain an assembly result after the second impurity removal. According to an embodiment of the invention, the second impurity removal further comprises removing duplicate assembly data.

A second alignment step S700: performing a second alignment of the second impurity-free assembly result with a known human genome sequence and a foreign gene sequence. According to an embodiment of the invention, the second alignment is performed using BWA.

Analysis step S800: Based on the second alignment result, the integration of the foreign gene in the human genome is determined. According to an embodiment of the present invention, determining the manner in which the foreign gene is integrated in the human genome based on the second alignment result further comprises: selecting and simultaneously aligning the known human genome sequence and the foreign gene sequence Assembly data, the assembly data includes human genome breakpoint information and foreign gene breakpoint information. According to an embodiment of the present invention, it may further be determined whether there is a replacement variation based on the human genome breakpoint information and the foreign gene breakpoint information; or determining, based on the human genome breakpoint information and the foreign gene breakpoint information, At least one of the length and type of foreign gene insertion in the human genome, for example, determining the length and type of insertion of at least a portion of the foreign gene in the human genome.

Referring now to Fig. 2, a system for determining the manner in which foreign genes are integrated in the human genome according to an embodiment of the present invention will be explained in detail by taking HBV as an example. As shown in FIG. 2, it specifically includes the following steps:

1. Acquisition and sequencing of nucleic acid integration sequences of pathogen genome fragments

Methods for obtaining a pathogen nucleic acid integration sequence include, but are not limited to, the following methods: A DNA fragment that may contain a pathogen genomic fragment is captured from a sample using a capture probe technique, and the resulting sequence is sequenced.

2. Remove PCR duplication, remove low quality sequencing data, and remove splice-containing sequencing data Strategy for removing PCR repeats: When the two sequences are identical, they are considered to be repeats. When one of the pair of PE sequencing data is duplicated, the pair of sequencing data is removed.

Strategy for removing low-quality sequencing data: When the number of bases with a sequencing quality value less than or equal to 5 in a sequencing data accounts for more than 50% of the total number of bases of the sequencing data, the sequencing data is considered to be low-quality sequencing data. . When one of the paired PE sequencing data is low quality, the pair of sequencing data is removed.

Strategies for removing sequence-containing sequencing data: When a piece of sequencing data contains a linker sequence, the sequence data is considered to contain linker sequencing data. When one of the paired PE sequencing data has sequenced sequencing data, the pair of sequencing data is removed.

3. Soap comparison, select the required sequencing data, calculate the comparison rate

The processed sequencing data were separately aligned to the human genome hgl9 and the pathogen genome fragment genome. Since the pathogen genome generally has multiple subtypes, the reference genome of the pathogen genome here generally needs to select the appropriate subtype according to the needs. After the alignment is completed, by analyzing the pairwise relationship between the two alignment results, sequencing data may be selected which may contain the integrated genome of the pathogen genome. The proportions of the useful sequences in the original sequencing data, and the ratio of the human genome alignment in the useful sequence to the genome of the pathogen genome fragment are calculated separately.

4. PER assembly

The sequencing data obtained in the third step, which may contain the integrated fragments of the pathogen genomic fragments, was subjected to PER assembly. PER refers to the assembly of pair end sequencing data. That is, each pair of PE sequencing data obtained by pair end sequencing is assembled according to the overlapping relationship between the sequences.

5. Remove duplicate sequences again

After assembly by PER, a collection of assembled sequences is obtained. This sequence set is deduplicated again. The strategy here is to use the deduplication strategy of SE sequencing data, ie when a sequence of sequencing data is duplicated, then the sequencing data is removed.

6. BWA heavy comparison, extract breakpoint information

By repeating the steps in the fifth step, a sequence set is obtained. This sequence set was then compared again to the human genome hgl9 and the pathogen genome using BWA software. By analyzing the results of the two alignments, sequences that are both comparable to the human genome hgl9 and the pathogen genome were selected. These sequences contain breakpoint information. The alignment of these sequences with the human genome hgl9 and the pathogen genome was analyzed separately, and the distribution of the pathogen integrated fragments on the human genome and the distribution on the pathogen genome were obtained.

The distribution here includes, but is not limited to, the alignment position, the number of sequencing data supporting the left endpoint of an insertion breakpoint, the number of sequencing data supporting the right endpoint of an insertion breakpoint, the total number of supported sequencing data, and the normalized amount of data. The number of supported sequencing data for the left endpoint, the number of sequencing data supported by the left endpoint, the total number of sequencing data supported after normalization, and the ID number of all supported sequencing data supporting an insertion breakpoint. The normalization strategy here is normalized based on the number of valid sequencing data.

The "sequencing data that supports the left endpoint of an insertion breakpoint" and "sequencing data that supports the right endpoint of an insertion breakpoint" are only relative to the insertion breakpoint in the human genome, for the pathogen genome, Then the breakpoint position is not inserted. 7. Check if there is a replacement category

The presence of a substitution type variation is examined by analyzing the intrinsic link between the human genome breakpoint and the pathogen genome breakpoint.

8. Calculate the length and type of the pathogen genomic insert

By analyzing the intrinsic link between the human genome breakpoint and the pathogen genomic breakpoint, we find the breakpoint information that can be used to calculate the pathogen genomic insert, and calculate the length and type of the pathogen genomic insert at this breakpoint.

9. Calculate the actual efficiency of capture

The result information of the integrated weight comparison and the initial sequence information are used to calculate the actual efficiency of the probe capture.

The method for determining the manner in which foreign genes are integrated in the human genome according to an embodiment of the present invention enables the discovery of precise pathogen genomic fragment insertion positions within the human genome. The method of determining the manner in which a foreign gene is integrated in the human genome according to an embodiment of the present invention can give some possible types of substitution, as well as the type of partial pathogen genomic insert. The method for determining the manner in which a foreign gene is integrated in the human genome according to an embodiment of the present invention is quick and convenient to use. Taking the starting data volume of 5G as an example, it can be analyzed within two days.

After extensive and intensive research, the present inventors have for the first time constructed a bioinformatics detection method for detecting the integration mode of a pathogen genome in a sample to be tested and its application, and specifically, the inventors obtained from a sequence capture technique. The sequences were aligned, screened, assembled, and compared, and a complete bioinformatics testing process was established. The present invention was completed on the basis of detecting the signals of the pathogen genome in the human genome integration mode using the detection procedure.

According to an embodiment of the present invention, the type of the sample that can be processed is not particularly limited as long as the nucleic acid sample is contained, and the type of the nucleic acid is not particularly limited, and may be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). ), preferably DNA. Those skilled in the art will appreciate that for RNA, it can be converted to cDNA having the corresponding sequence by conventional means for subsequent detection and analysis. According to an embodiment of the present invention, the source of the sample is not particularly limited. According to an example of the present invention, a cancer tissue sample can be used as a test sample, whereby a DNA sequence of a pathogen genomic insert can be extracted therefrom, and the insertion of the pathogen genomic fragment can be detected and analyzed. Examples of samples that may be used in accordance with embodiments of the invention include, but are not limited to, patient plasma, cancer tissue cells, paracancerous tissue cells.

In one embodiment of the invention, DNA library preparation is required prior to application of the probe for sequence capture, and methods for preparing sample libraries are well known to those skilled in the art. The term "DNA library preparation" refers to disrupting a target fragment of a genome to obtain a mixture of DNA fragments of a certain size. According to an embodiment of the present invention, the method and apparatus for capturing a specific sequence from a sample are also not particularly limited and can be carried out using a commercially available probe. In the present invention, the sequencing sequence refers to a sequence fragment output by the sequencer, that is, sequencing data (reads). In one embodiment of the invention, the DNA fragments used for sequencing are captured by a particular probe. The probe must be pure and not affected by other different sequence nucleic acids. Typical probes are cloned DNA sequences or DNA obtained by PCR amplification, synthetic oligonucleotides or RNA obtained after cloning and cloning DNA sequences in vitro, and can also be used as probes. The length of the probe may range from 20 to 500 mers, preferably from 50 to 300 mers, more preferably from 250 mers. Probe design and synthesis methods are those skilled in the art As is well known, synthetic probes can be used to synthesize probes or to use commercially available probes.

In the present invention, the sequencing sequence obtained from the sample can be carried out by a sequencing method, which can be performed by any sequencing method, including but not limited to the dideoxy chain termination method; preferably a high-throughput sequencing method, including but not limited to Second generation sequencing technology or single molecule sequencing technology. The second generation sequencing platform described in the present invention (Metzker ML. Sequencing technologies-the next generation. Nat Rev Genet. 2010 Jan; ll(l): 31-46) includes but is not limited to Illumina (eg GA series, HiSeq series) , Life Technologies (such as SOLID series, semiconductor sequencing series, etc.) and Roche (such as GS series) and other companies provide sequencing platforms.

The types of sequencing described in the present invention include, but are not limited to, Pair-end sequencing, and the sequencing length includes, but is not limited to, 100 bp. In one embodiment of the present invention, the sequencing platform is Illumina/Solexa, and the sequencing type is Pair-end sequencing, and a 100 bp DNA sequence molecule having a bidirectional positional relationship is obtained.

In the present invention, the main library length of the sequencing sequence should be less than 200 bp. This will allow for sufficient assembly success to ensure sufficient data for subsequent studies. In some embodiments of the invention, the plasma sample has a sequencing yield of about 5G and the tissue cell sample has a sequencing data volume of about 1G. The larger the amount of data, the more comprehensive the information that can be detected.

Using the methods of the present invention, pathogen genomic insertion signals can be determined by data generated by next generation sequencing techniques, including but not limited to location, genotype, number, and length.

In the present invention, the human genome sequence for performing SOAP2 alignment and BWA alignment is the human genome reference sequence of version 37 (hgl9; NCBI Build 37) in the NCBI database.

In some embodiments of the invention, the pathogen reference sequence for performing SOAP2 alignment and BWA alignment is selected from 23 genomic sequences of the 8 subtypes of the pathogen.

In the present invention, the alignment includes PER pre-assembly alignment and PER post-assembly alignment, wherein the PER pre-assembly alignment is a mismatch of 5 bases. The PER pre-assembly sequence alignment can be performed by any sequence alignment program, such as the Short Oligonucleotide Analysis Package (SOAP) and BWA alignments available to those skilled in the art (Burrows-Wheeler Aligner 0.5. 8c (rl536)), the sequencing sequence is aligned with the reference genome sequence, and the sequencing data is classified according to the alignment of the sequencing data with the pathogen genome and the human genome. The sequence alignment can be performed using default parameters provided by the program, or the parameters can be selected by those skilled in the art as needed. In one embodiment of the invention, the comparison software employed is SOAPaligner/soap2. Another alignment is post-assembly alignment of the PER. Sequence alignment after PER assembly can be performed by any sequence alignment program that can be set to allow a sufficient length of gap. The sequence alignment can be performed using default parameters provided by the program, or the parameters can be selected by those skilled in the art as needed. For example, the B WAS W parameter in the BWA alignment (Burrows-Wheeler Aligner) available to those skilled in the art is performed, and the alignment position is determined by comparison.

In some embodiments of the invention, for step 3, the conditions for extracting sequencing data that may contain information on the pathogen genomic insert (the following conditions are met) are:

1) In a pair of PE sequencing data, in the case of only 5 mismatches, one can compare with the human genome and the other can compare with the pathogen genome;

2)—In the case of PE sequencing data, in the case where only 5 mismatches are allowed, one can compare with the human genome, and the other Cannot compare to any reference sequence;

3)—In the case of PE sequencing data, in the case of only 5 mismatches, one can compare with the pathogen genome, and the other cannot match any reference sequence;

4) In a pair of PE sequencing data, in the case where only 5 mismatches are allowed, neither of them can match any reference sequence. In some embodiments of the present invention, for the Soap alignment step in Figure 2, the required sequencing data is selected, and the comparison ratio is calculated: This step outputs a file including but not limited to the following items: Yield, comparison rate , pollution rate, proportion of valid sequencing data. Effective sequencing data refers to the sequencing data remaining after the original down-sequencing data is removed from the contaminated sequencing data. The contaminated sequencing data referred to herein refers to PCR repeat sequencing data, including linker sequencing data, and low quality sequencing data.

In some embodiments of the present invention, for the Soap alignment step of Figure 2, the required sequencing data is selected and the alignment ratio is calculated, wherein the insert sd value of the Soap alignment is set to 30. This ensures the largest possible sequencing data utilization. In combination with Table 1 below, the number of sequencing data set in a certain grid is V _num , and the human genome comparison rate is alnRate. The pathogen transcript rate is alnRate _virus , and V _all = Vi+Vz+Vs+Vs +Vs+V +Vg+Vg then the formula for calculating the ratio is alnRate _vims = ( V1+V2+V4+V5+V3+V6 ) IV _all , alnRate ( V1+V2+V4+V5+V7+V8 ) I Van

In some embodiments of the present invention, for the soap alignment step in Figure 2, the required sequencing data is selected to calculate the alignment ratio: conditions for extracting sequencing data that may contain information on the pathogen genomic insert (the following conditions are satisfied) Yes 1) In a pair of PE sequencing data, in the case of only 5 mismatches, the strip can be compared to the human genome, and the other can be compared to the pathogen genome; 2) - for PE sequencing data, only 5 In the case of a mismatch, the strip can be compared to the human genome, and the other cannot be compared to any reference sequence; 3) - For PE sequencing data, in the case of only 5 mismatches, one can compare with the pathogen genome, One cannot compare with any reference sequence; 4) In a pair of PE sequencing data, in the case where only 5 mismatches are allowed, neither of them can match any reference sequence.

In some embodiments of the present invention, "calculating useful sequences in raw sequencing data" in the SOAP alignment step means removing PE sequencing data containing PCR repeats, PE sequencing data containing low quality sequencing data, and Sequence of AP sequencing data containing adaptor. 5 6 8 9 of Table 1 below is a sequence selected in some embodiments of the present invention. The rows in Table 1 indicate the alignment of the sequence with the human genome, PE indicates that a pair of PE sequencing data can be compared to the upper reference sequence within the set insert length; SE indicates that only one pair of PE sequencing data Can be compared to the reference sequence, or both can be compared to the reference sequence, but the alignment position is not within the set insertion length; Unmap indicates that a pair of PE sequencing data can not match the reference sequence at all.

Table 1. Sequencing data classification

In the present invention, the step of removing the repeated sequence again in Fig. 2: although the PCR repeating operation in the PE sequencing data has been performed in the first impurity removal step 2, the filtering is not thorough. of. Because some of the overlapping parts of the sequence may have mismatches, but they can still be assembled, it may result in the same sequence after assembly.

In some embodiments of the present invention, for the BWA re-alignment step, the breakpoint information is extracted: BWA 0.5.8c (rl536) The parameters used in the heavy comparison are parameters BWASW that are suitable for long sequence alignment and support high fault tolerance alignment. . It uses the heuristic Smith-Waterman-like algorithm to search for high-scoring alignment locations. The parameters used in the comparison completely use the default parameter values of the software. For details, please refer to http://bio-bwa.sourceforge.net/bwa.shtml o It uses the heuristic Smith-Waterman-like algorithm to search for high scores. Compare the positions.

In the BWA weight comparison step in the present invention, the breakpoint information is extracted: after the BWA weight comparison is completed, the comparison result needs to be processed to extract the breakpoint information. At this time, the selection of the alignment result for each sequence needs to meet the following conditions:

1) Simultaneously compare the human genome with the pathogen genome.

2) Regardless of the type of reference genome, the length of the sequence aligned with the reference sequence must be greater than or equal to 30 bp.

3) Regardless of the type of reference genome alignment, the sequence length of the insert portion may be greater than or equal to

5bp

In some embodiments of the invention, for the BWA re-alignment step, the breakpoint information is extracted: the BWA alignment is a basic strategy of truncation and truncation. For example, when the first half of a piece of sequencing data is inferior to the reference sequence, the comparison software will directly cut off the portion of the sequenced data that is not comparable, and then continue to compare.

In some embodiments of the invention, for the BWA heavy comparison step, the breakpoint information is extracted:

When extracting breakpoint information from the comparison results, several situations may be encountered, and the following are specifically listed and the processing method of the present invention is given. The cases given below are all possible when the sequence is aligned with the human genome. The investigator should understand that the alignment position given by the comparison software BWA is the left end position of the sequence. Suppose the alignment position of the comparison software is p

1) The upstream part of the sequence is the pathogen genome sequence, the downstream part is the human DNA sequence, and there are no other types of bases. At this time, the pathogen sequence insertion position of the pathogen should be compared with the alignment position given by the software.

2) - The upstream part of the sequence is the human DNA sequence, and the downstream part is the pathogen genome sequence, and there are no other types of bases. At this time, the insertion position of the pathogen genome sequence fragment should be compared with the alignment position given by the software. Assume that the length of the DNA sequence of the upstream human is X, and the insertion position of the pathogen genome sequence fragment should be taken as p+x 3) The upstream and downstream of one sequence are human genome sequences, and the middle part is the pathogen genome insert, assuming the upstream person the length of the DNA sequence portion of the human DNA sequence of the downstream portion of the length X of _χ τ, time pathogen genomic insert position to be taken as p + x _t

4) Both the upstream and downstream of a sequence are pathogen genomic sequences, and the middle part is the human genome sequence. This sequence carries the signal of the insertion of the two pathogen genomic fragments into the integration point, so it can be extracted from the alignment result of this sequence. Two insertion positions. Assume that the length of the DNA sequence of the upstream human is y _t, the length of the DNA portion of the downstream human is y _T , and the length of the human genome of the middle part is y _† , and the insertion position of the genomic fragment of the pathogen should be taken as p and p+y _† In one embodiment of the present invention, for the BWA heavy comparison step, the breakpoint information is extracted: the output file of the step gives the number of left support sequencing data, the number of right support sequencing data, the total number of supported sequencing data, and the corresponding return The number of sequencing data after the ization. Here are some explanations for these items. The number of sequencing data supported by the left is the number of sequencing data that supports the left endpoint of a certain insertion breakpoint. Specifically, the number of sequences upstream of the pathogen genomic insert relative to the aligned reference sequence. The right supports the number of sequencing data, that is, the number of sequencing data that supports the right endpoint of a certain insertion breakpoint. Specifically, the number of sequences downstream of the pathogen genomic insert relative to the aligned reference sequence. The total number of supported sequencing data is equal to the sum of the number of left support sequences and the number of right support sequences. The number of normalized left support sequences. The number of left support sequencing data before the normalization is V _L , and the useful logarithm of the sequencing data is b M pairs, then the number of left support sequences after normalization is Vi7b. The number of normalized support sequences after normalization. Before normalization, the number of right support sequencing data is a, and the useful sequencing data log is V _r M pairs, then the number of left support sequences after normalization is VJb. The normalized total number of support sequences is the sum of the number of normalized left support sequences and the number of normalized right support sequences.

It is necessary to perform the de-repetitive sequence operation on the sequenced data in the obtained output file, because the de-repeat operation of steps 2 and 5 does not take into account the case where the reverse complement sequence or the repeat sequence is also considered. Therefore, it is necessary to perform the left and next screening of the sequence in the obtained result, and remove the redundant sequence to obtain an optimal result.

In some embodiments of the present invention, the step of checking whether there is a substitution category: the specific method is when two breakpoint information on the human genome appears to have only left support sequencing data or only right support sequencing data, then two The sequence between the breakpoint positions is the human genome sequence in which the substitution occurs. The alignment positions of the left support sequencing data and the right support sequence data on the pathogen genome were found separately, and the sequence between the two aligned positions found was the pathogen genome sequence in which the substitution occurred. In the present invention, all displacements will be output in accordance with a random combination of eligible breakpoints on the human genome.

In some embodiments of the invention, the step of calculating the length and type of the pathogen genomic insert: the specific method is to find the left-end support sequence and the right-end support sequence of a pathogen genomic insert, and then find the support sequences at the two ends, respectively. In the alignment position on the pathogen genome, the sequence between the two positions is the insert.

The step of calculating the actual efficiency for the calculation of the present invention: The specific method is to calculate the number of sequences participating in the BWA heavy alignment, which is comparable to the upper human genome hgl9 and the pathogen genome, and is denoted as A. The logarithm of the PE sequencing data useful in the original sequencing data is recorded as B. The effective capture efficiency is calculated as A/B.

In the present invention, the last breakpoint information that can be found includes breakpoint information of the pathogen genome fragment on the human genome and breakpoint information of the pathogen genome fragment on the pathogen genome.

In the present invention, the biological information analysis process involved can be theoretically applied to signal detection in the case where all pathogen genetic material DNA or RNA is inserted into the human genome. A system for determining the way in which foreign genes are integrated in human factors

According to still another aspect of the present invention, the present invention provides a system for determining the manner in which a foreign gene is integrated in a human due group. Referring to FIG. 3, the system includes a capture device 100, a sequencing device 200, a first impurity removal device 300, and a first alignment device. 400, assembly device 500, second impurity removal device 600, second comparison device 700, and analysis device 800. Among them, referring to FIG. 3, these devices are sequentially connected in the process flow.

According to an embodiment of the present invention, the capture device 100 utilizes a capture probe to capture a DNA fragment that may contain an integration of a foreign gene fragment from a human genomic nucleic acid sample. According to an embodiment of the present invention, the type of exogenous gene that can be analyzed using the method of the present invention is particularly limited. As long as it can be integrated with the human genome, and its gene sequence can be obtained or already known. According to an embodiment of the invention, the foreign gene that can be studied is the pathogen genome. Further, according to a specific example of the present invention, the pathogen is HBV. Thereby, the integration of pathogens such as HBV with the human genome can be effectively analyzed.

According to an embodiment of the present invention, the sequencing device 200 performs sequencing on the captured DNA fragments to obtain sequencing results composed of a plurality of sequencing data. According to an embodiment of the present invention, the manner of sequencing the captured DNA fragments is not particularly limited. According to an embodiment of the invention, sequencing is performed by a second generation sequencing platform. According to an embodiment of the present invention, the whole genome sequencing library can be sequenced using at least one selected from the group consisting of Hiseq2000, SOLiD, 454, and a single molecule sequencing device. Thereby, the efficiency of determining the aneuploidy of single cell chromosomes can be further improved by utilizing the characteristics of high-throughput and deep sequencing of these sequencing devices. Of course, those skilled in the art will appreciate that other sequencing methods and devices can be used for whole genome sequencing, such as third generation sequencing techniques, as well as more advanced sequencing techniques that may be developed in the future. According to an embodiment of the present invention, the length of the sequencing data obtained by whole genome sequencing is not particularly limited. According to an embodiment of the present invention, it is preferable that the sequencing length is lOObp, whereby the analysis effect can be further improved.

According to an embodiment of the present invention, the first impurity removing device 300 performs the first impurity removal on the sequencing result to obtain the sequencing result of the first impurity removal. According to an embodiment of the present invention, the type of the first impurity is performed, and is not particularly limited. For example, the first impurity may further include at least one of removing PCR repeats, removing low-quality sequencing data, and removing linker-containing sequencing data. Thereby, the analysis efficiency can be further improved.

According to an embodiment of the present invention, the first comparison device 400 firstly compares the first impurity-sequencing result with a known human genome sequence and a foreign gene sequence to obtain a possible integration of the foreign gene. Sequencing data for the fragment. According to an embodiment of the invention, the first alignment can be performed using SOAP. Thereby, the analysis efficiency can be further improved.

According to an embodiment of the present invention, the assembly device 500 assembles the obtained sequencing data which may contain the foreign gene integration fragment to obtain an assembly result, which is composed of a plurality of assembly data. According to an embodiment of the invention, the assembly is performed by based on an overlapping relationship between the sequencing data.

According to an embodiment of the present invention, the second impurity removing device 600 performs second impurity removal on the assembly result to obtain an assembly result of the second impurity removal. According to an embodiment of the invention, the second impurity removal further comprises removing duplicate assembly data.

According to an embodiment of the invention, the second alignment device 700 performs a second alignment of the second impurity-free assembly result with a known human genomic sequence and a foreign gene sequence. According to an embodiment of the invention, the second alignment is performed using BWA.

The analysis device 800 determines the manner in which the foreign gene is integrated in the human genome based on the second alignment result. According to an embodiment of the present invention, determining the manner in which the foreign gene is integrated in the human genome based on the second alignment result further comprises selecting and simultaneously aligning the known human genome sequence and the assembly of the foreign gene sequence Data, the assembly data includes human genome breakpoint information and foreign gene breakpoint information. According to an embodiment of the present invention, it may further be determined whether there is a replacement variation based on the human genome breakpoint information and the foreign gene breakpoint information; or determining, based on the human genome breakpoint information and the foreign gene breakpoint information, At least one of the length and type of foreign gene insertion in the human genome, for example, determining the length and type of insertion of at least a portion of the foreign gene in the human genome.

It should be noted that those skilled in the art will appreciate that the features and advantages of the methods described above for determining the manner in which foreign genes are integrated in the human genome are also suitable for systems for determining the manner in which foreign genes are integrated in the human genome. The description is convenient and will not be described in detail. Computer readable shield

In yet another aspect of the invention, the invention provides a computer readable medium. According to an embodiment of the invention, instructions are stored on the computer readable medium, the instructions being adapted to be executed by the processor to determine how the foreign gene is integrated in the human genome by the following steps: performing a first impurity removal on the sequencing result In order to obtain the sequencing result of the first impurity removal; the first alignment result of the first impurity is first aligned with the known human genome sequence and the foreign gene sequence, so as to obtain an integration fragment which may contain the foreign gene Sequencing data; assembling the obtained sequencing data, which may contain the integrated fragment of the foreign gene, to obtain an assembly result, the assembly result being composed of a plurality of assembly data; performing second impurity removal on the assembly result to obtain Performing a second impurity-free assembly result; and performing a second alignment of the second impurity-free assembly result with a known human genome sequence and a foreign gene sequence, and determining based on the second alignment result The manner in which the foreign gene is integrated in the human genome, wherein the sequencing result is obtained by the following : capturing a DNA fragment that may contain an integration of a foreign gene fragment from a human genomic nucleic acid sample using a capture probe; sequencing the captured DNA fragment to obtain a sequencing result composed of a plurality of sequencing data. The computer readable medium can be used to efficiently determine the manner in which foreign genes, such as pathogen genomes, are integrated in the human genome.

For the purposes of this specification, a "computer-readable medium" can be any apparatus that can contain, store, communicate, propagate, or transport the program for use by the instruction execution system, apparatus, or device, or in conjunction with the instruction execution system, apparatus, or device. More specific examples (non-exhaustive list) of computer readable media include the following: electrical connections (electronic devices) having one or more wires, portable computer disk cartridges (magnetic devices), random access memory (RAM), Read only memory (ROM), erasable editable read only memory (EPROM or flash memory), fiber optic devices, and portable compact disk read only memory (CDROM). Furthermore, the computer readable medium may even be a paper or other suitable medium on which the program can be printed, as it may be optically scanned, for example by paper or other medium, followed by editing, interpretation or, if appropriate, other suitable The method proceeds to obtain the program electronically and then store it in computer memory.

It should be understood that portions of the invention may be implemented in hardware, software, firmware or a combination thereof. In the above-described embodiments, multiple steps or methods may be implemented in software or firmware stored in a memory and executed by a suitable instruction execution system. For example, if implemented in hardware, as in another embodiment, it can be implemented by any of the following techniques or combinations thereof known in the art: having logic gates for implementing logic functions on data signals Discrete logic circuits, application specific integrated circuits with suitable combinational logic gates, programmable gate arrays (PGAs), field programmable gate arrays (FPGAs), and the like.

A person skilled in the art can understand that all or part of the steps carried by the method of the foregoing embodiment can be completed by a program to instruct related hardware, and the program can be stored in a computer readable storage medium. In execution, one or a combination of the steps of the method embodiments is included.

In addition, each functional unit in each embodiment of the present invention may be integrated into one processing module, or each unit may exist physically separately, or two or more units may be integrated into one module. The above integrated modules can be implemented in the form of hardware or in the form of software functional modules. The integrated modules, if implemented in the form of software functional modules and sold or used as separate products, may also be stored in a computer readable storage medium.

The above-mentioned storage medium may be a read only memory, a magnetic disk or an optical disk or the like.

It should be noted that those skilled in the art can understand that the features and advantages of the method for determining the manner in which the foreign gene is integrated in the human genome described above are also suitable for the computer readable medium, which is convenient for description and will not be described in detail. . The solution of the present invention will be explained below in conjunction with the embodiments. Those skilled in the art will appreciate that the following examples are merely illustrative of the invention and are not to be considered as limiting the scope of the invention. In the examples, the specific techniques or conditions are not indicated, according to the techniques or conditions described in the literature in the field (for example, refer to J. Sambrook et al., Huang Peitang et al., Molecular Cloning Experimental Guide, Third Edition, Science Press) or in accordance with the product manual. The reagents or instruments used are not specified by the manufacturer, and are conventional products that are commercially available, for example, from Illumina. Example 1

Sample library preparation

Sample source

The source of the sample is a patient's liver cancer tissue, and the patient's liver cancer tissue has genome-wide sequencing information and breakpoint information found by genome-wide data.

2. Pre-experiment

The pre-risk section includes the following steps:

(1) Extract DNA.

(2) Preparing a sample library

The library was constructed according to Illumina's Paired-End Sample Preparation Guide. The genomic DNA was disrupted with Covaris s2, the ends were filled with repair, the end was added with A, the linker was added, and the fragment added to the adaptor was PCR. Sample library.

(3) Preparation of HBV capture probe

Design probes for b and c types of HBV. The length of each probe is 60 bp, and the design principle is that the overlap between adjacent probes is 55 bp in length. Specifically, the steps for preparing the HB V probe are as follows: Design primers, PCR reaction, PCR purification and electrophoresis detection, PCR product fragmentation, fragmentation product electrophoresis detection, and probe storage. (4) Hybridization and sequencing of HBV capture probes with sample libraries

The HB V capture probe was hybridized with the sample library using Nimblegen's hybridization platform, hybridized, eluted, PCR amplified, and the PCR product was sequenced. Then, sequencing was performed using the Hiseq 2000 sequencing platform, in which the sequencing was performed in accordance with the c-Bot and Hiseq 2000 (PE sequencing) specifications officially published by IUumina/Solexa. The main sequence library was 170 bp in length, the sequencing sequence was lOObp in length, and the sequencing yield was lG bp.

3. Bioinformatics analysis

(1) Remove PCR repeats, remove low-quality sequencing data, and remove sequencing-containing sequencing data

After getting the data, the PCR is repeated for these data, the low quality sequencing data is removed, and the sequencing data containing the linker is removed.

Strategy for removing PCR repeats: When the two sequences are identical, they are considered to be repeats. When one of the paired PE sequencing data is duplicated, the pair of sequencing data is removed.

Strategy for removing low-quality sequencing data: When the number of bases with a sequencing quality value less than or equal to 5 in a sequencing data accounts for more than 50% of the total number of bases of the sequencing data, the sequencing data is considered to be low-quality sequencing data. When a pair

When one of the PE sequencing data is low quality, the pair of sequencing data is removed.

Strategy for removing sequencing data containing linkers: When a sequence of linker data contains a linker sequence, the sequenced data is considered to be sequenced data containing the linker. When one of the paired PE sequencing data is sequencing data containing the linker, the pair of sequencing data is removed.

4. Soap comparison, select the required sequencing data, calculate the comparison rate

The processed sequencing data were aligned to the human genome hgl9 and the HBV genome, respectively. Because the HBV virus has multiple subtypes, the HBV genome here includes 23 subtypes (AB014381.1, AB032431.1, AB033554.1, AB036910.1, AB064310.1, AF090842.1, AF100309.1, AF160501. 1, AF223965.1, AF405706.1, AY090454.1, AY090457.1, AY090460.1, AY123041.1, D00329.1, M32138.1, X02763.1, X04615.1, X51970.1, X65259.1, Genomic sequence of X69798.1, X75657.1, X85254.1). After the comparison is completed, by analyzing the pairwise relationship between the two alignment results, the sequencing data that may contain the viral integration fragment is selected. The proportions of useful sequences in the original sequencing data were calculated separately, as well as the human genome alignment rate and the HBV genome alignment rate in the useful sequences. The parameters of the Soap comparison: -m l38 -x l98 -p 8 -140 -v 5 -r l. Table 2 below is the data quality report, Table 3 is the classification result of the sequencing data by the preliminary soap comparison result, and Table 4 is the comparison rate statistics.

Table 2. Data Quality Report

GC content 48.78; 49.08

Proportion of sequencing data with linker 0.07%

Low quality sequencing data ratio 6.32%

PCR repetition rate 5.14%

Effective data ratio 88.47% Table 3. Sequencing data Jiugong grid classification results

Table 4. Comparison rate statistics

5. PER assembly

The sequencing data obtained in the third step, which may contain the viral integration fragment, was subjected to PER assembly. PER refers to the assembly of pair end sequencing data. That is, each pair of PE sequencing data obtained by pair end sequencing is assembled according to the overlapping relationship between the sequences. Assembly success rate is 94.33%

6. Remove duplicate sequences again

After assembly by PER, a collection of assembled sequences is obtained. This sequence set is deduplicated again. The strategy here is to use the deduplication strategy of SE sequencing data, ie when a sequence of sequencing data is duplicated, then the sequencing data is removed. As a result, 1.823% of the repeats were removed, leaving 850,696 available sequences after assembly.

7. BWA heavy comparison, extract breakpoint information

By repeating the steps in the fifth step, a sequence set is obtained. This sequence set was then again aligned to the human genome hgl9 and HBV viral genomes using BWA software. By analyzing the results of the two alignments, sequences were selected that simultaneously corresponded to the human genome hgl9 and HBV viral genomes. These sequences contain breakpoint information. The alignment of these sequences with the human genome hgl9 and HBV viral genomes was separately analyzed, and the integration of the HBV virus on the human genome and the distribution on the HBV viral genome were obtained. Finally, 33 breakpoints were found, of which the number of breakpoints exceeding the threshold was 8. A de-duplication operation is performed on the obtained result to obtain a final result. As shown in Table 5 below, the most prominent viral insertion site for insertion of the HBV virus found in the present invention into the human genome hgl9 is shown. Table 5. HBV virus insertion breakpoint information

8. Check if there is a replacement category

The presence of a substitution type variation is examined by analyzing the intrinsic link between the human genome breakpoint and the HBV viral genome breakpoint. The specific method is that when the two breakpoint information on the human genome appears to be within 500 bp, and both breakpoints have only the left end supporting the sequencing data or only the right end supporting the sequencing data, then the two breakpoints are likely to be A situation has occurred in the replacement. Find the HBV viral genome alignment information for the two breakpoints and determine the type and location of the replacement. Table 6 below shows the seven replacement cases found by the present invention.

Table 6. Types of substitution

9. Calculate the length and type of HBV inserts

By analyzing the internal links between the human genome breakpoints and HBV viral genome breakpoints can be calculated to find the HBV viral insert the breakpoint information, calculates the breakpoint in this portion of HBV insert length and another type ¹ J . Specifically, a left-end support sequence and a right-end support sequence of a viral insert are found, and then the alignment positions of the two-end sequences on the HBV viral genome are found, and the sequence between the two positions is an insert. Only one insertion breakpoint is found here to find out the type of insert. Table 7 below is the type of insert found in the present invention. Table 7. HBV virus insert type

10. Calculate the effective efficiency of capture

The result information of the integrated weight comparison and the initial sequence information are used to calculate the actual efficiency of the probe capture. The specific method is to calculate the number of sequences in the BWA heavy comparison that can compare with the human genome hgl9 and the HBV viral genome, and record it as A. The logarithm of the PE sequencing data useful in the raw sequencing data is recorded as B. Then, the effective capture efficiency is calculated as A/B. The effective capture efficiency calculated here is 0.0001059. Industrial applicability

The method, system and computer readable medium of the present invention for determining the manner in which a foreign gene is integrated in the human genome can be effectively used to determine the manner in which a foreign gene, such as a pathogen genome, is integrated in the human genome. In the description of the present specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" and the like means a specific feature described in connection with the embodiment or example. A structure, material or feature is included in at least one embodiment or example of the invention. In the present specification, the schematic representation of the above terms does not necessarily mean the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in a suitable manner in any one or more embodiments or examples.

While the embodiments of the present invention have been shown and described, the embodiments of the invention may The scope of the invention is defined by the claims and their equivalents.

Claims

claims

1. A method for determining how foreign genes are integrated in the human genome, which is characterized by: using capture probes to capture DNA fragments that may contain integrated foreign gene fragments from human genome nucleic acid samples; targeting the captured DNA Sequencing the fragments to obtain a sequencing result composed of multiple sequencing data; performing a first impurity removal on the sequencing result to obtain a sequencing result that has undergone the first impurity removal;

Perform a first comparison of the first-removed sequencing results with known human genome sequences and exogenous gene sequences to obtain sequencing data that may contain exogenous gene integration fragments;

Assemble the obtained sequencing data that may contain exogenous gene integration fragments to obtain an assembly result, which is composed of multiple assembly data;

Performing a second impurity removal on the assembly result to obtain an assembly result that has undergone the second impurity removal; and

Perform a second alignment of the assembly result that has undergone the second cleanup with known human genome sequences and exogenous gene sequences, and based on the second alignment result, determine the position of the exogenous gene in the human genome Integrated approach.

2. The method according to claim 1, characterized in that the exogenous gene is a pathogen genome.

3. The method according to claim 2, characterized in that the pathogen is HBV.

4. The method of claim 1, wherein the sequencing is performed using a second-generation sequencing platform.

5. The method of claim 1, wherein the first impurity removal further includes at least one of removing PCR repeats, removing low-quality sequencing data, and removing sequencing data containing adapters.

6. The method of claim 1, wherein the first comparison is performed using SOAP.

7. The method of claim 1, wherein the assembly is performed based on overlapping relationships between sequencing data.

8. The method of claim 1, wherein the second impurity removal further includes removing duplicate assembly data.

9. The method of claim 1, wherein the second comparison is performed using BWA.

10. The method according to claim 9, characterized in that, based on the second comparison result, determining the integration mode of the foreign gene in the human genome further includes:

Select assembly data that can simultaneously compare known human genome sequences and foreign gene sequences. The assembly data contains human genome breakpoint information and foreign gene breakpoint information.

11. The method according to claim 10, further comprising:

Based on the human genome breakpoint information and exogenous gene breakpoint information, determine whether there is a substitution mutation; or based on the human genome breakpoint information and exogenous gene breakpoint information, determine the length and type of exogenous gene insertion in the human genome At least one.

12. A system for determining the integration mode of foreign genes in the human genome, characterized by comprising: a capture device, the capture device is suitable for using capture probes to capture the integration of foreign gene fragments that may contain from human genome nucleic acid samples DNA fragments;

A sequencing device, the sequencing device is connected to the capture device and is adapted to perform sequencing on the captured DNA fragments. Sequencing, in order to obtain sequencing results composed of multiple sequencing data;

A first impurity removal device, the first impurity removal device is connected to the sequencing device, and is adapted to perform a first impurity removal on the sequencing result, so as to obtain a sequencing result that has undergone the first impurity removal;

A first comparison device, the first comparison device is connected to the first impurity removal device, and is suitable for comparing the sequencing results after the first impurity removal with known human genome sequences and foreign gene sequences. The first comparison is to obtain sequencing data that may contain integrated fragments of foreign genes;

Assembly device, the assembly device is connected to the first comparison device, and is suitable for assembling the obtained sequencing data that may contain exogenous gene integration fragments, so as to obtain an assembly result, the assembly result is composed of multiple assembly Data composition;

a second impurity removal device, the second impurity removal device is connected to the assembly device, and is adapted to perform a second impurity removal on the assembly result, so as to obtain an assembly result that has undergone the second impurity removal;

A second alignment device, the second alignment device is connected to the second impurity removal device, and is suitable for comparing the assembly result after the second impurity removal with known human genome sequences and foreign gene sequences. second comparison; and

An analysis device, the analysis device is adapted to determine the integration mode of the foreign gene in the human genome based on the second comparison result.

13. The system according to claim 12, characterized in that the sequencing device is a second-generation sequencing platform.

14. The system according to claim 12, characterized in that the first impurity removal device further includes at least one of the following:

Units suitable for removing PCR repeats;

Units suitable for removing low-quality sequencing data; and

A unit suitable for removing sequencing data containing adapters.

15. The system according to claim 12, characterized in that the first comparison device is adapted to perform comparison using SOAP.

16. The system according to claim 12, wherein the assembling device is adapted to assemble based on overlapping relationships between sequencing data.

17. The system according to claim 12, wherein the second impurity removal device further includes a unit adapted to remove duplicate assembly data.

18. The system according to claim 12, characterized in that the second comparison device is adapted to perform comparison using BWA.

19. The system according to claim 18, characterized in that the analysis device is adapted to select assembly data that can simultaneously compare known human genome sequences and foreign gene sequences, and the assembly data includes human genome fragments. point information and foreign gene breakpoint information.

20. The system according to claim 19, characterized in that the analysis device is suitable for:

21. A computer-readable medium, characterized in that instructions are stored on the computer-readable medium, and the instructions are suitable for execution by a processor to determine the integration mode of foreign genes in the human genome through the following steps:

Perform the first impurity removal on the sequencing results to obtain the sequencing results that have undergone the first impurity removal;

Perform a second alignment of the assembly result that has undergone the second cleanup with known human genome sequences and exogenous gene sequences, and based on the second alignment result, determine the position of the exogenous gene in the human genome Integration method,

Wherein, the sequencing results are obtained through the following steps:

Use capture probes to capture DNA fragments that may contain integrated exogenous gene fragments from human genome nucleic acid samples; sequence the captured DNA fragments to obtain sequencing results consisting of multiple sequencing data.

22. The computer-readable medium according to claim 21, wherein the foreign gene is a pathogen genome.

23. The computer-readable medium according to claim 22, wherein the pathogen is HBV.

24. The computer-readable medium according to claim 21, wherein the sequencing is performed by a second-generation sequencing platform.

25. The computer-readable medium according to claim 21, wherein the first impurity removal further includes at least one of removing PCR repeats, removing low-quality sequencing data, and removing sequencing data containing adapters.

26. The computer-readable medium of claim 21, wherein the first comparison is performed using SOAP.

27. The computer-readable medium according to claim 21, wherein the assembly is performed based on overlapping relationships between sequencing data.

28. The computer-readable medium according to claim 21, wherein the second removal of impurities further includes removing duplicate assembly data.

29. The computer-readable medium of claim 21, wherein the second comparison is performed using BWA.

30. The computer-readable medium according to claim 29, characterized in that, based on the second comparison result, determining the integration manner of the foreign gene in the human genome further includes:

31. The computer-readable medium according to claim 30, further comprising: