CN110527714A - For detecting HPV in the method for the integration site of host genome - Google Patents
For detecting HPV in the method for the integration site of host genome Download PDFInfo
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Abstract
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.
Description
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.
Embodiment
The present embodiment is used for exemplary illustration the method for the present invention.
One, sample information
The fresh cancerous tissue of an example IIIb Cervix Squamous Cell cancer is selected to carry out HPV integration detection as sample.
Two, experimental procedure
1, extracting genome DNA
The extraction of genomic DNA uses salting out method, and respectively using Nanodrop 2000, Qubit mode it is quantitative and
1.0% Ago-Gel carries out clip size Quality Control.Genomic DNA yield is greater than 3 μ g, and segment is greater than 5kb.
2, DNA is interrupted
Sample interrupts method and interrupts pipe using microTUBE-50, interrupts 50 μ l of volume, interrupts parameter using Peak
incident Power(W):75；Duty Factor:5%；Cycles Per Burst:200.Genomic DNA is broken into master
Peak different length 800bp segment, 2.0% Ago-Gel run colloid inspection, the next hybrid capture of qualified sample and
Enriching step.
3, HPV probe hybridizes
Target area is with reference to genome sequence according to the HPV gene order of the website NCBI, and target acquistion region is that HPV is complete
Genome area carries out successional probe design in full-length genome region.DNA spy is designed and synthesized according to HPV full-length genome
Needle (Intergrated DNA Technolaogy), referring to xGen hybridization capture of DNA
Libraries (Intergrated DNA Technolaogy) operation instructions carry out hybrid capture to virus sequence, obtain mesh
Mark sequence is simultaneously enriched with using PCR.PCR product after enrichment uses Agilent High Sensitivity DNA 2100
The sample of chip detected magnitude and abundance, satisfactory amount of DNA and quality for testing in next step.
4, other purposes section amplification method
The sample that capture requires cannot be reached for starting amount of DNA, be enriched with using the method for amplification template.Specific plan
Slightly: a. interrupts DNA for purpose fragment length, single-ended jointing, a plurality of primer and list covered with HPV genome sequence
The amplification of end connector universal primer carries out amplified production to build library in next step after purification；B. for the height being had been reported in database
Hair integrates hotspot location design primer, and sequence of breakpoints is included in amplified production region, and amplified production purifies quality inspection, meets item
Library is built in the progress of part in next step.
5, the building of sequencing library
5.1 ends DNA are repaired, and are added " A " and are purified
Reaction system is as shown in table 1, and configured reaction mixture is shaken and is mixed, reaction condition: 20 DEG C, 30min；65
DEG C, 30min；4 DEG C of holdings.After reaction, product purification is carried out using 60 μ l (1 ×) AMPure XP magnetic beads, recycling DNA is molten
In 25 μ l nuclease free waters.
Repair system in -1 end of table
The connection of 5.2 bar codes
Bar code coupled reaction system is as shown in table 2, and configured reaction mixture is shaken and is mixed, 25 DEG C of incubations
15min.Product purification is carried out using 50 μ l (1 ×) AMPure XP magnetic beads, recycling DNA is dissolved in 26 μ l nuclease free waters.
- 2 bar code linked system of table
The connection of 5.3 connectors
Connector interfaces system is as shown in table 3, and configured reaction mixture is shaken and is mixed, and is incubated at room temperature 10min.It uses
40 μ l (0.4 ×) AMPure XP magnetic beads carry out product purification, and the washing of 140 μ l connector magnetic bead association reaction liquid (ABB) is then added
2 times, recycling DNA is dissolved in 15 μ l elution buffers (ELB).Product is quantitative using Qubit mode.
- 3 connector interfaces system of table
6, upper machine sequencing
It is sequenced using nano-pore sequencing instrument (Oxford Nanopore Technology MinION).
6.1 prepare chip preparation solution
Chip preparation liquid system is as shown in table 4, prepares a sequence testing chip, and open chip and prepare mouth, first eliminates inner wall
Then the mixed liquor in 800 μ l tables 4, static 5min is added in bubble.
- 4 chip of table prepares liquid system
6.2 pre- sequencing solution preparations and loading
Loading system is as shown in table 5, while keeping chip preparation hole to open, well is opened, first slowly by 200 μ l
Chip preparation solution injects chip and prepares hole, then solution is sequenced in 75 μ l in advance and adding mouth is added dropwise.Finally, close adding mouth and
Chip prepares mouth, starts to be sequenced, make target area average sequencing depth reach 10000 × more than.
- 5 loading system of table
Three, data are analyzed
7.1 data Quality Controls
Original lower machine data FASTQ file Quality Control according to Oxford Nanopore Technologies company EPI2ME
Agent Barcoding analysis process carries out Quality Control, and the smallest qscore value is 7.
7.2 sequence alignment
Clean sequence is compared with HPV and human genome sequence using Blat analysis process, is obtained containing than confrontation
Measure score value, aligned sequences location information etc. as a result, alignment parameters are blat-stepSize=5-repMatch=2253-
MinScore=20-minIdentity=0-noHead refence_database input.fa output.psl.
7.3 score values and the output of consistency result
According to the formula of UCSC, the score value and consistency of comparison result are calculated, it is best to choose HPV and human comparison respectively
As a result, the resultant output listing that two results are grouped together；
The judgement of 7.4 HPV integration sequence positions
Retain and had not only compared HPV sequence in list but also compared the sequencing reading length of upper human genome sequence and compare HPV and people
Positional relationship of the sequence in sequencing reading length, positional distance of two kinds of aligned sequences in the sequence are to be spaced or be overlapped 10
The case where within base, it is believed that the sequence is HPV integration sequence；Merge identical sequence of breakpoints and count sequence of breakpoints number, there is 5
It is correct HPV integration site that the above sequence of item, which is detected the judgement with the identical breakpoint,.
7.5 functional annotation
Function on annovar software progress genome is passed through to the breakpoint location on the HPV and human genome in the sequence
It can annotation；
8, result verification
Its recall rate is measured to known integration site using the present invention, in these sites of detection, is sequenced by two generations
Method and Sanger are verified and are calculated its verifying rate.
Four, experimental result
It is known to 17 detections/19 (89.5%) using the recall rate of method of the invention to known integration site.In addition, should
Method has also detected other unknown integration sites 39, in these sites of detection, has 15 to be verified by two generation sequencing approaches,
In addition 13 sites have been extracted and have carried out Sanger verifying, wherein 13 sites are all verified as correct integration site, verifying
Rate is the 100% of sampling, and the primer information of 13 integration sites is shown in Fig. 1, and as shown in Figure 2, Sanger sequencing is disconnected for PCR amplification result
Point result is shown in Fig. 3, shown in 4.17 known breakpoints of detection are shown in Fig. 5.In conclusion using the analysis process to known to an example
59 integration sites are found altogether in the integration site detection result of the positive sample of HPV integration, wherein 45 are tested by other methods
Card, remaining 14 sites are not verified, and total verifying rate in site is 76.3%.The HPV breakpoint identified according to distinct methods
Number relatively Venn figure is as shown in Figure 6.
Although the present invention has been described with reference to exemplary embodiments, however, it is to be understood that the present invention is not limited to disclosed
Exemplary implementation scheme.It, can be to the exemplary reality of description of the invention without departing substantially from the scope or spirit of the invention
The scheme of applying does a variety of adjustment or variation.The scope of the claims should be based on widest explanation to cover all modifications and equivalent structure
With function.
Claims (10)
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.
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| CN111020019A (en) * | 2020-03-06 | 2020-04-17 | 元码基因科技（北京）股份有限公司 | Method for gene fusion detection based on nanopore technology |
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