阅读说明：本技术 用于检测hpv在宿主基因组的整合位点的方法 (For detecting HPV in the method for the integration site of host genome ) 是由 孟博 田埂 王伟伟 董如一 杨文娟 于 2019-09-06 设计创作，主要内容包括：本发明公开一种用于检测HPV在宿主基因组的整合位点的方法,包括以下步骤：从来自受试者的生物样本中提取宿主基因组DNA,并将其打断至主峰为800bp-4kb的片段；利用探针组对得到的片段中进行第一检测获得目标序列；对获得的目标序列进行预处理以提高检测灵敏度；和基于电信号测序技术在不进行PCR扩增的情况下对目标序列进行第二检测,从而确认HPV在宿主基因组的整合位点。本发明的方法能够快速、及时分析HPV病毒存在于宿主细胞内的状态判断,具有显著的临床应用优势,应用本方法在无需错误纠正分析的情况下即可更有效地判断HPV的整合信息。(The present invention discloses a kind of method for detecting integration site of the HPV in host genome, comprising the following steps: extracts host genome DNA from from the biological sample of subject, and is interrupted the segment for being 800bp-4kb to main peak；Target sequence is obtained to the first detection is carried out in obtained segment using probe groups；The target sequence of acquisition is pre-processed to improve detection sensitivity；The second detection is carried out to target sequence without PCR amplification with based on electric signal sequencing technologies, to confirm HPV in the integration site of host genome.Method of the invention can analyze the state judgement that HPV viruse is present in host cell quickly, in time, have significant clinical application advantage, can more effectively judge the integration information of HPV without error correcting analysis using this method.)

1. a kind of method for detecting integration site of the HPV in host genome, which comprises the following steps:

(1) from from the biological sample of subject extract host genome DNA, and by the host genome DNA interrupt to Main peak is the segment of 800bp-4kb；

(2) the first detection is carried out in step (1) obtained segment using probe groups and obtains target sequence, wherein the spy Needle group is made of multiple probes, and each probe respectively include can with the sequence of the specific region selective cross of HPV genome, The target sequence includes the sequence from the sequence of HPV gene and from host genome；

(3) target sequence obtained in step (2) is pre-processed to obtain pretreated target sequence to improve detection spirit Sensitivity；With

(4) the second detection is carried out to target sequence without PCR amplification based on electric signal sequencing, to confirm Integration site of the HPV in host genome.

2. the method according to claim 1 for detecting integration site of the HPV in host genome, which is characterized in that The biological sample is cervical exfoliated cell or cervical cancer tissues.

3. the method according to claim 1 for detecting integration site of the HPV in host genome, which is characterized in that Each probe in the probe groups is designed as the continuity region for HPV full-length genome.

4. the method according to claim 1 for detecting integration site of the HPV in host genome, which is characterized in that Pretreatment in the step (3) includes being enriched with to the target sequence.

5. the method according to claim 1 for detecting integration site of the HPV in host genome, which is characterized in that Pretreatment in the step (3) include the target sequence both ends add the first connector, then by using with it is described The primer of first connector selective cross carries out PCR amplification.

6. the method according to claim 1 for detecting integration site of the HPV in host genome, which is characterized in that Second detection of the step (4) includes adding A in the end of pretreated target sequence, is then sequentially connected bar code and the Two connectors, followed by nano-pore sequencing.

7. the method according to claim 6 for detecting integration site of the HPV in host genome, which is characterized in that The depth of the nano-pore sequencing is 5000 × to 10000 ×.

8. a kind of method for detecting integration site of the HPV in host genome, which comprises the following steps:

(1) from from the biological sample of subject extract host genome DNA, and by the host genome DNA interrupt to Main peak is the segment of 800bp-4kb；

(2) the segment one end connect third connector, using the primer sets being made of multiple specific primers and with it is described The universal primer of third connector selective cross expands to obtain target sequence, wherein the multiple specific primer separately designs For that can hybridize with the continuity sequence selectivity in HPV genome, so that the primer sets be made to cover entire HPV genome sequence Column；With

(3) target sequence is detected without PCR amplification based on electric signal sequencing, to confirm that HPV exists The integration site of host genome.

9. a kind of method for detecting integration site of the HPV in host genome, which comprises the following steps:

(1) from from the biological sample of subject extract host genome DNA, and by the host genome DNA interrupt to Main peak is the segment of 800bp-4kb；

(2) third connector is connected in one end of the segment, using the primer sets of multiple specific primers composition and with described the The universal primer of three connector selective cross expands to obtain target sequence, wherein the multiple specific primer separately design for It can be with a kind of gene order selective cross of integration site side known to HPV；With

(3) target sequence is detected without PCR amplification based on electric signal sequencing, to confirm that HPV exists The integration site of host genome.

10. -9 described in any item methods for detecting integration site of the HPV in host genome according to claim 1, It is characterized in that, the confirmation HPV further comprises data analysis step in the integration site of host genome comprising:

Quality Control is carried out to raw sequencing data, noise data is removed, obtains data to be analyzed；

The data to be analyzed are compared respectively with HPV genome sequence and host genome sequence, obtain containing comparison The result of quality score, aligned sequences location information；

According to the formula of UCSC, the score value and consistency of comparison result are calculated, HPV is chosen respectively and host compares best knot Fruit, the resultant output listing that two results are grouped together；

Retain list in not only with HPV genome sequence again with the sequencing reading length that host genome sequence compares completely and compared with the two Positional relationship, when positional distance of two kinds of aligned sequences in the sequence be spaced or overlapping 10 bases when, judgement should Sequence is HPV integration sequence；

Merge identical sequence of breakpoints and count sequence of breakpoints number, when there are 5 or more sequences to be detected with the identical breakpoint, then sentences Break as correct HPV integration site.

Technical field

The present invention relates to the detections of viral integration sites, more particularly to for detecting HPV in the integration of host genome The method in site.

Background technique

It is the significant process event for causing cancer and occurring that HPV viruse, which is integrated into human genome, at present to the understanding of the process Also than relatively limited, more and more researches show that HPV integration is the potential molecular marker as molecular diagnosis and individualized treatment Object, and very valuable prognostic indicator.The research method of HPV integration is applied based on two generation sequencing technologies more at present Probe catching method obtains.For example, CN107739761A discloses the high-flux sequence detection side of a kind of HPV parting and integration Method.This method chooses the gene of current HPV hypotype, in conjunction with second generation high throughput sequencing technologies, more fully detection patient infection The type of HPV overcomes the difficulties such as traditional detection method accuracy rate is low, false positive is high, poor repeatability, rate of missed diagnosis height.In molecule Diagnostic field, most direct and specific technology is gene sequencing, and second generation high throughput sequencing technologies than mostly using classics at present Sanger sequencing approach have inspection is more flux-intensive, sequencing speed faster, that accuracy is higher, cost is lower and information content is richer etc. is excellent Point.This method can carry out accurately typing to high-risk HPV and low risk HPV under the help of second generation high throughput sequencing technologies And its integration that human genome whether occurs is detected, accurately individuation is carried out to tester and is assessed, prevention lesion occurs Risk, thus the generation of pre- preventing tumor.Although being all directed to short-movie section based on the capture technique comparative maturity of two generations sequencing The probe of DNA captures or target fragment amplification, and two generation sequencing reading lengths are shorter and sensitivity is too high, easily lead to false positive As a result, and two generation methods only breakpoint location is detected, the integrated structure of long sequence is difficult to identify.

Recently, three generations's sequencing increasingly highlights the characteristics of its length reads long and quick detection cause of disease, but for viral integrase position The detection of point is also more rare, and the error rate of three generations's sequencing result is higher, and generally acknowledged error rate is 10-15% at present, gives As a result credibility brings problem, therefore generally requires the step of increasing error correction for the analysis of three generations's sequencing data, from And improve the accuracy and confidence level of sequencing result.Although the method for existing many error corrections, such as according to two generations and three generations Sequencing result three generations is corrected, or three generations's result is subjected to multiple self-correcting to reduce error rate, but due to The result difference of correction software and the limitation of user's selective power cause significantly to limit for its application.

Summary of the invention

To solve at least partly technical problem in the prior art, the present invention provides a kind of for detecting HPV in host's base Because of the method for the integration site of group.Specifically, the present invention includes the following contents.

The first aspect of the present invention provides a kind of method for detecting integration site of the HPV in host genome, packet Include following steps:

(1) host genome DNA is extracted from from the biological sample of subject, and the host genome DNA is beaten The segment broken to main peak for 800bp-4kb；

(2) the first detection is carried out in step (1) obtained segment using probe groups and obtains target sequence, wherein institute It states probe groups to be made of multiple probes, and respectively include can be with the specific region selective cross of HPV genome for each probe Sequence, the target sequence include the sequence from the sequence of HPV gene and from host genome；

(3) target sequence obtained in step (2) is pre-processed to obtain pretreated target sequence to improve inspection Survey sensitivity；With

(4) the second detection is carried out to target sequence without PCR amplification based on electric signal sequencing, thus really HPV is recognized in the integration site of host genome.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the biology sample This is cervical exfoliated cell or cervical cancer tissues.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the probe groups In each probe be designed as the continuity region for HPV full-length genome.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the step (3) pretreatment in includes being enriched with to the target sequence.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the step (3) pretreatment in includes adding the first connector at the both ends of the target sequence, then by selecting with first connector Property hybridization primer carry out PCR amplification.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the step (4) the second detection includes adding A in the end of pretreated target sequence, is then sequentially connected bar code, followed by Nano-pore sequencing.

Preferably, the method according to the present invention for detecting integration site of the HPV in host genome, the nano-pore The depth of sequencing is 5000 × to 10000 ×.

The second aspect of the present invention, provide it is another for detecting HPV in the method for the integration site of host genome, The following steps are included:

(1) host genome DNA is extracted from from the biological sample of subject, and the host genome DNA is beaten The segment broken to main peak for 800bp-4kb；

(2) the segment one end connect third connector, using the primer sets being made of multiple specific primers and with The universal primer of the third connector selective cross expands to obtain target sequence, wherein the multiple specific primer difference It is designed to hybridize with the continuity sequence selectivity in HPV genome, so that the primer sets be made to cover entire HPV gene Group sequence；With

(3) target sequence is detected without PCR amplification based on electric signal sequencing, to confirm Integration site of the HPV in host genome.

The third aspect of the present invention, provide it is another for detecting HPV in the method for the integration site of host genome, The following steps are included:

(1) host genome DNA is extracted from from the biological sample of subject, and the host genome DNA is beaten The segment broken to main peak for 800bp-4kb；

(2) the segment one end connect third connector, using multiple specific primers composition primer sets and with institute The universal primer for stating third connector selective cross expands to obtain target sequence, wherein the multiple specific primer is set respectively Being calculated as can be with a kind of known gene order selective cross of HPV integration site side；With

(3) target sequence is detected without PCR amplification based on electric signal sequencing, to confirm Integration site of the HPV in host genome.

Further, the method according to the present invention for detecting integration site of the HPV in host genome, the confirmation HPV further comprises data analysis step in the integration site of host genome comprising: matter is carried out to raw sequencing data Control removes noise data, obtains data to be analyzed；Make the data to be analyzed and HPV genome sequence and host genome sequence Column are compared respectively, obtain containing comparison quality score, the result of aligned sequences location information；According to the formula of UCSC, meter The score value and consistency of comparison result are calculated, HPV is chosen respectively and host compares best as a result, two results are grouped together Resultant output listing；Retain the sequencing not only compared completely with HPV genome sequence but also with host genome sequence in list to read The positional relationship for growing and comparing the two, when positional distance of two kinds of aligned sequences in the sequence is to be spaced or be overlapped 10 alkali When base, judge that the sequence is HPV integration sequence；Merge identical sequence of breakpoints and count sequence of breakpoints number, there are 5 or more sequences When being detected with the identical breakpoint, then it is judged as correct HPV integration site.

The present invention carries out Preliminary detection using the segment that interrupts using probe or primer pair host genome for the first time, then right The target sequence that Preliminary detection obtains formulated optimization builds library and analysis strategy, thus establish it is a set of from experimental method establish to The method for accurately carrying out HPV viruse breakpoint analysis can be quick by being not necessarily to the bioinformatic analysis process of error correction Integration site of the HPV viruse in human genome is accurately identified, needs to provide new strategy to meet scientific research and clinic.

Detailed description of the invention

The primer and sequence information of exemplary authentication breakpoint in Fig. 1 the method for the present invention.

New breakpoint PCR amplification result schematic diagram in Fig. 2 the method for the present invention.The figure is the spy for the method for the present invention detection DNA fragmentation product after determining the people of integration site design and the primer PCR amplification of HPV viruse sequence carries out 1% Ago-Gel The result of electrophoresis.Wherein, each letter of A-O respectively represents 13 different breakpoint locations.The figure illustrates each pair of primer The integration site sequence that this method sequencing obtains all successfully is amplified.

Breakpoint result schematic diagram is sequenced based on Sanger in Fig. 3 the method for the present invention.Each letter of A-O respectively represents 13 A different breakpoint location.As a result display is the generation Sanger method sequencing result of Fig. 2 amplified production, these sequencing results in The sequence for having groups of people and HPV is proved after the website NCBI blast compares, and the sequencing result of position and this method is always. Overall length sequencing information is in Fig. 1.

Fig. 4 is the 13 breakpoint location information verified by Sanger method.

Fig. 5 is 17 known breakpoints of detection.

Fig. 6 is to compare Venn figure based on the HPV breakpoint number that distinct methods identify.Wherein, Nanopore result is this hair Bright method qualification result, NGS are two generation sequencing approach qualification results, and Paper is the known breakpoint result delivered.

Specific embodiment

The existing various exemplary embodiment that the present invention will be described in detail, the detailed description are not considered as to limit of the invention System, and it is understood as the more detailed description to certain aspects of the invention, characteristic and embodiment.

It should be understood that it is to describe special embodiment that heretofore described term, which is only, it is not intended to limit this hair It is bright.In addition, for the numberical range in the present invention, it is thus understood that specifically disclose the range upper and lower bound and they it Between each median.Median and any other statement value in any statement value or stated ranges or in the range Lesser range is also included in the present invention each of between interior median.These small range of upper and lower bounds can be independent Ground includes or excludes in range.

Unless otherwise stated, all technical and scientific terms used herein has the routine in field of the present invention The normally understood identical meanings of technical staff.Although the present invention only describes preferred method and material, of the invention Implement or also can be used and similar or equivalent any method and material described herein in testing.The institute mentioned in this specification There is document to be incorporated by reference into, to disclosure and description method relevant to the document and/or material.It is incorporated to any When document conflicts, it is subject to the content of this specification.

The method that the determination multi-pass of integration site to virus in the genome crosses Sanger sequencing and the sequencing of two generations at present, On the one hand, the sequencing of two generations has certain advantage, but it is read length and is difficult to detect to the viral integrase structure of long sequence, confidence level It is lower.On the other hand, nanometer sequencing technologies need multiple self-correcting to reduce its error rate.In view of above-mentioned two o'clock, the present invention By the way that the study found that being enriched with the integration site sequence of HPV viruse using the design of multiple groups probe, the sequencing and analysis in library are built in optimization Strategy, establish it is a set of from experimental method to carry out HPV viruse breakpoint analysis method, and pass through without mistake check and correction biology Bioinformatics analysis process can rapidly and accurately identify integration site of the HPV viruse in human genome, can be to meet section It grinds and clinic needs to provide new strategy.

Method of the invention generally comprises following steps: (one) extracts genomic DNA and obtains the step of interrupting segment； (2) the step of obtaining target sequence；(3) integration site of the confirmation HPV in host genome.The following detailed description of each step Suddenly.

Step (1):

Step (one) of the invention is to extract genomic DNA and obtain the step of interrupting segment.In exemplary implementation scheme In comprising host genome DNA is extracted from from the biological sample of subject, and being interrupted to main peak is 800bp- The segment of 4kb, as step (1).

Subject of the invention is generally mammal or the mankind, is preferably people.It is relevant that biological sample is generally uterine neck Sample, including cervical exfoliated cell or cervical cancer tissues.Host genome is the genome corresponding to subject, is generally people's base Because of group.

Genome of the invention, which interrupts mode, can be used any means known.These means can refer to known textbook, such as Public publications such as " Molecular Cloning:A Laboratory guide " fourth edition of Cold SpringHarbor etc..The length of the segment interrupted needs the main peak to be 800bp-4kb, preferably 1kb-4kb, more preferable 1.5kb-3.5kb, such as 2.5kb.

Step (2):

Step (two) of the invention is the step of obtaining target sequence from obtained segment.Wherein target sequence refers to Comprising the sequence from HPV gene and from the sequence of host genome.Sequence from HPV gene can be located at mesh 5 ' the ends for marking sequence can also be located at its 3 ' end.This is not particularly limited.It similarly, can position from the sequence of HPV gene In 5 ' ends of target sequence, its 3 ' end can also be located at.

In certain embodiments, step of the invention (two) includes the following steps:

(2) target sequence is obtained to the first detection is carried out in obtained segment using probe groups, wherein probe groups are by multiple Probe composition, and respectively include can be with the sequence of the specific region selective cross of HPV genome for each probe；(3) to acquisition Target sequence pre-processed to improve detection sensitivity.

The purpose of first detecting step of the invention is that the host genome of the gene containing HPV is obtained by specific probe groups Segment, i.e., containing the target sequence of integration site.Probe groups of the invention are the probe groups of specially designed multiple probe compositions It closes.Preferably, each probe in probe groups is to the different zones of HPV full-length genome, and these different zones are continuity region, To cover HPV full-length genome.Preferably, probe groups for HPV full-length genome coverage be 2 ×.Here HPV full genome Group is the gene regions for including coding carcinogenic protein important factor comprising early gene area, late gene area and long control region.

In step (3), i.e., the target sequence of acquisition is pre-processed to improve in detection sensitivity, pretreatment may include The process that target sequence is enriched with.The first connector is added at the both ends that pretreatment may additionally include target sequence respectively, then By carrying out PCR amplification using the primer of at least part selective cross with the first connector.The sequence length of first connector Generally 6-50nt, preferably 10-30nt, more preferable 10-20nt.As long as the sequence of the first connector can with host genome and Sequence in HPV genome distinguishes, then is not particularly limited.First joint sequence may include known array and random sequence.Example Such as, it is known that sequence can be 6 to 13 continuous bases, and random sequence can be 1 to 13 continuous base.For example, first Joint sequence can be as shown in TATGGGCAGTCGT.Connector known in the art can be used in first connector.In a kind of exemplary implementation In scheme, the first connector used is preferably suitable for connector used in A-T connecting-type library, more preferably, such as The blocker sequence of Intergrated DNA TechnologiesUniversal Blocker-TS Mix sequence and envelope The Human Cot-1DNA of genome repetitive sequence is closed, is then connect in such a way that both ends connect with host DNA segment, then PCR amplification is carried out with the primer of the first connector selective cross.PCR product after enrichment uses Agilent High Sensitivity DNA2100 chip detects its size and abundance.

In certain embodiments, step of the invention (two) include by the combination of specific primer and universal primer come Obtain target sequence.Specific primer of the invention is generally multiple, thus forms primer sets.Universal primer of the invention is general It is one.Universal primer is applied in combination with primer sets.

Specific primer of the invention separately design for can with the specific region selective cross in HPV genome, it is more The corresponding specific region composition continuity region of a specific primer or sequence, so that primer sets be made to cover entire HPV genome Sequence.

Universal primer is introduced thus, and the present invention needs to connect third connector in the one end for interrupting segment.The sequence of third connector Column length is generally 6-50nt, preferably 10-30nt, more preferable 10-20nt.As long as the sequence of third connector can be with host gene Sequence in group and HPV genome distinguishes, then is not particularly limited.Third joint sequence may include known array and stochastic ordering Column.For example, as it is known that sequence can be 6 to 13 continuous bases, random sequence can be 1 to 13 continuous base.For example, Third joint sequence can be as shown in TATGGGCAGTCGT.Connector known in the art can be used in third connector.Third connector can be with It is identical as above-mentioned first connector, it can also be different.It should be noted that different from the first connector, third connector can only be connected to The single-ended of segment is interrupted, and is not connectable to both ends.

In certain embodiments, specific primer design of the invention be can be with a kind of integration site known to HPV The gene order selective cross of side, multiple specific primers then can be with the bases of multiple integration sites side known to HPV Because sequence selectivity hybridizes, the information of specific integration site is obtained so as to specificity.Known integration site is usually Refer to and frequently-occurring integrates hot spot.For example, non-homology recombination is high-incidence to integrate hotspot location, generation area includes fragile site, dystopy Broken site, transcriptionally active site.

Step (3):

Step (three) of the invention is to confirm HPV in the integration site of host genome.It specifically, may include based on electricity Signal sequencing carries out the second detection to target sequence without PCR amplification, to confirm HPV in host genome Integration site, i.e. step (4).In exemplary arrangement, step (4) includes adding to the target sequence end obtained after the first detection Then A is sequentially connected bar code and the second connector, carry out nano-pore sequencing.It should be noted that sequencing process here is not Include the steps that PCR, in an exemplary embodiment, sequencing depth be 10000 ×, to reach to HPV integration site sequence Depth detection.Specifically, it repairs or adds using the end NEBNext in the end DNA of the biological sample obtained by the first detection DA tail modular reagent box, it is preferable that repaired using such as NEBNext End Repair/dA-tailed reagent to carry out the end DNA The addition of multiple and dA tail adds primary bar code after purification and connects, and the second connector is added after being further purified is connected to preparing The end DNA, it is preferable that the second connector is the Ligation Sequencing of Oxford Nanopore Technology company The sequence measuring joints provided in Kit kit, progress magnetic bead is pure after connecting DNA product the second connector of connection of bar code after purification Change, then carries out nano-pore sequencing.Magnetic beads for purifying step is carried out using known method, in an exemplary embodiment, is used AMPure XP magnetic bead carries out product purification.Primary bar code and the second connector can be synthesized or are purchased from by known method known Commodity, such as use no PCR bar code connector or Barcode modular reagent box, it is preferable that use Oxford Nanopore The primary bar code and bar code connector provided in the Ligation Sequencing Kit kit of Technology company, Adapt to the nano-pore sequencing machine and subsequent bioinformatics process analysis in the method for the present invention without PCR.

Step (three) of the invention further includes the steps that data processing or analysis.Known nano-pore sequencing result error rate compared with Height, error rate is 10%-15% at present.Error correction at present is carried out by analysis software.In view of the difference of software output result Different and user's selective power limitation, and the reorganizing research for being applied to virus is very rare.Bioinformatics through the invention Analysis process, the qualification requirement based on integration sequence merge two kinds of sequence alignment methods, and the parameter of optimal screening breakpoint, from And the speed and accuracy of analysis can be significantly improved, find the viral integration sites information that accurately can verify that.Specifically, originally The analysis of biological information process of invention includes: to carry out Quality Control to raw sequencing data, removes noise data, obtains number to be analyzed According to Quality Control process is not specially limited, in an exemplary embodiment, public according to Oxford Nanopore Technologies The EPI2ME agent Barcoding analysis process of department carries out Quality Control, and the smallest qscore value is 7；Make data to be analyzed with HPV genome sequence and host genome sequence are compared respectively, obtain containing comparison quality score, aligned sequences position letter The result of breath, it is preferable that its reduced parameter is blat-stepSize=5-repMatch=2253-minScore=20- MinIdentity=0-noHead refence_database input.fa output.psl；According to the formula of UCSC, meter The score value and consistency of comparison result are calculated, HPV is chosen respectively and host compares best as a result, two results are grouped together Resultant output listing；Retain the sequencing not only compared completely with HPV genome sequence but also with host genome sequence in list to read The positional relationship for growing and comparing the two, when positional distance of two kinds of aligned sequences in the sequence is to be spaced or be overlapped 10 alkali When base, judge that the sequence is HPV integration sequence；Merge identical sequence of breakpoints and count sequence of breakpoints number, there are 5 or more sequences When being detected with the identical breakpoint, then it is judged as correct HPV integration site；To on the HPV and human genome in the sequence Breakpoint location by annovar software carry out genome on functional annotation.It should be noted that the present invention use with As analysis foundation, it is suitable for the integration site of other DNA integration virus in host to detect analysis for HPV integration site detection Process.

Further, by the Library development flow and analysis method of optimization described above, precise Identification of the present invention is multiple out HPV integrates the breakpoint location in positive sample, and is verified by Sanger method, and set identification HPV disease is as a result demonstrated The strategy of poison is highly effective.