CN105543380A - Method and device for detecting gene fusion - Google Patents
Method and device for detecting gene fusion Download PDFInfo
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Abstract
The invention discloses a method and device for detecting gene fusion. The method comprises the following steps that 1, DNA of a sample to be detected is extracted, and then a joint is disconnected or connected; 2, an Agilent probe customized according to a target gene is used for capturing a drive gene in fusion, to be detected, of the target gene through a target area capturing method to obtain a target DNA fragment; 3, a high-throughput sequencing method is used for sequencing the target DNA fragment to obtain a sequence of a fusion form; 4, after low-quality sequences are filtered out, the sequence of the fusion form is detected. By the application of the technical scheme, the target area capturing technology is used for capturing the gene fused often, the gene fused often is just sequenced, and therefore the sequencing cost is reduced greatly. In addition, a target area is utilized for capturing fusion to propel single-end fusion signals of the gene, a database is combined, and therefore fusion detection sensitivity of gene data captured in the target area can be high.
Description
Technical field
The present invention relates to field of bioinformatics, in particular to a kind of method and the device that detect gene fusion.
Background technology
Unexpected, heritable variation phenomenon (genemutation) that transgenation refers to that genomic DNA molecule occurs.From molecular level, transgenation refers to the change that gene base pair composition structurally occurs or puts in order.Although gene is very stable, accurately can copy oneself when cell fission, this stability is relative.Gene also can become another kind of new existence form from original existence form flip-flop under certain conditions, and be exactly on a site, occurred suddenly a new gene, instead of original gene, this gene is called mutator gene.So also just suddenly there is the new proterties that ancestors never have in the performance of offspring.
Transgenation is one of important factor of organic evolution, so research transgenation also has biological significance widely except the theory significance of itself.Transgenation provides saltant type for genetics research, for breeding work provides material, so its practical significance of also having scientific research and producing.
Some transgenations are because the variation of karyomit(e) recurring structure is formed.Under the impact of natural condition or human factor, the structure variation that karyomit(e) occurs mainly contains: disappearance, repeat, inversion and transposition, wherein, gene fusion is also the one of karyomit(e) recurring structure variation.So-called fusion gene, refers to and is joined end to end the coding region of two or more gene, is placed under same set of regulating and controlling sequence (comprising promotor, enhanser, ribosome binding sequence, terminator etc.) control, the mosaic gene of formation.
At present, in the order-checking of two generations, conventional gene fusion detection method is based on the high-flux sequence on the DNA level of full-length genome, uses fusion gene two end signal simultaneously, adds statistical test, judges the generation of merging.But genome sequencing can not have the high order-checking degree of depth, and the method that target area is caught generally only designs and catch one end of common fusion gene, and the other end not designing probe cause there is no signal, this two problems all can cause the sensitivity of fusion detection very low.
Summary of the invention
The present invention aims to provide a kind of method and the device that detect gene fusion, and to find fusion sequence from DNA level by high throughput sequencing technologies, the two generations order-checking solving based target areas captured is for the low technical problem of fusion detection sensitivity.
To achieve these goals, according to an aspect of the present invention, a kind of method detecting gene fusion is provided.The method comprises the following steps: S1, extracts the DNA of sample to be detected, then interrupts, jointing; S2, the driving gene in the fusion to be checked of the method target acquisition gene using the Agilent probe customized according to target gene to utilize target area to catch and then obtain target DNA fragments; S3, is checked order to target DNA fragments by the method for high-flux sequence, obtains the sequence of fusion form; S4, after filtering out low-quality sequence, the sequence of detection fusion form; S4 specifically comprises: S41, the sequence of the fusion form utilizing comparison software to be obtained by S3 and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sorts according to the position of comparison, then sets up the indexed file of comparison result; S42, the comparison result of the sequence merging form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing soft sequence of blocking sequence, the soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, require to include 1) block sequence at least 4 containing non-equal is soft, and can consensus sequence be mated; 2) the soft number of blocking sequence cannot mating consensus sequence contained is less than the soft number of blocking sequence can mating consensus sequence; With 3) consensus sequence is greater than 30nt; S43, detect known fusion form: the comparison result analyzing S42, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And detect new fusion form: the comparison result analyzing S42, if not there is the record merged with first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.
Further, what the comparison software in S41 adopted is BWA-mem comparison software, and the software setting up the employing of comparison result indexed file is samtools software.
Further, the use picard software in S42 removes the tumor-necrosis factor glycoproteins that PCR produces.
Further, many sequences in S42 are 4 sequences.
Further, in S43, the aligned sequences that homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.
According to another aspect of the present invention, a kind of device detecting gene fusion is provided.This device comprises: DNA processing module, for extracting the DNA of sample to be detected, then interrupts, jointing; Gene trap module, the driving gene in the fusion to be checked of the method target acquisition gene utilizing target area to catch for using the Agilent probe customized according to target gene and then obtain target DNA fragments; Sequencer module, checks order to target DNA fragments for the method by high-flux sequence, obtains the sequence of fusion form; Detection module, after filtering out low-quality sequence, the sequence of detection fusion form; Detection module specifically comprises: sequence alignment submodule, the sequence of the fusion form obtained by detection module for utilizing comparison software and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sort according to the position of comparison, then set up the indexed file of comparison result; Sequence screening submodule, for the comparison result of the sequence merging form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing soft sequence of blocking sequence, the soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, require to include 1) block sequence at least 4 containing non-equal is soft, and can consensus sequence be mated; 2) the soft number of blocking sequence cannot mating consensus sequence contained is less than the soft number of blocking sequence can mating consensus sequence; With 3) consensus sequence is greater than 30nt (bases longs); Detect known fusion form submodule: for the comparison result of analytical sequence screening submodule, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And detect new fusion form submodule: for the comparison result of analytical sequence screening submodule, if not there is the record merged with first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.
Further, what the comparison software in sequence screening submodule adopted is BWA-mem comparison software, and the software setting up the employing of comparison result indexed file is samtools software.
Further, the use picard software in sequence screening submodule removes the tumor-necrosis factor glycoproteins that PCR produces.
Further, many sequences in sequence screening submodule are 4 sequences.
Further, detect in new fusion form submodule, the aligned sequences that homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.
Apply technical scheme of the present invention, combining target areas captured technology, high throughput sequencing technologies and fusion sequence information analysis, provide the flow process of a set of highly sensitive detection fusion from DNA.Wherein, by the gene of catching normal generation fusion of target area capture technique, only the normal gene occurring to merge is checked order, greatly reduce order-checking cost; In addition, single-ended fusion signal that lower fusion orders about gene adds that database can detect very high sensitivity to the fusion of the gene data that target area is caught to utilize target area to catch.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the schematic flow sheet of the method detecting gene fusion according to an embodiment of the invention; And
Fig. 2 show assemble according to an embodiment of the invention soft block the step of sequence with judge the schematic diagram of confidence level.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
At present, in the order-checking of two generations, conventional gene fusion detection method is based on the high-flux sequence on the DNA level of full-length genome, uses fusion gene two end signal simultaneously, adds statistical test, judges the generation of merging.But genome sequencing can not have the high order-checking degree of depth, and the method that target area is caught generally only designs and catch one end of common fusion gene, and the other end not designing probe cause there is no signal, this two problems all can cause the sensitivity of fusion detection very low.For above-mentioned deficiency of the prior art, the invention provides following technical scheme.
According to a kind of typical embodiment of the present invention, provide a kind of method detecting gene fusion.The method comprises the following steps: S1, extracts the DNA of sample to be detected, then interrupts, jointing; S2, the driving gene in the fusion to be checked of the method target acquisition gene using the Agilent probe customized according to target gene to utilize target area to catch and then obtain target DNA fragments; S3, is checked order to target DNA fragments by the method for high-flux sequence, obtains the sequence of fusion form; S4, after filtering out low-quality sequence, the sequence of detection fusion form; S4 specifically comprises: S41, the sequence of the fusion form utilizing comparison software to be obtained by S3 and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sorts according to the position of comparison, then sets up the indexed file of comparison result; S42, the comparison result of the sequence merging form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing soft sequence of blocking sequence, the soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, require to include 1) block sequence at least 4 containing non-equal is soft, and can consensus sequence be mated; 2) the soft number of blocking sequence cannot mating consensus sequence contained is less than the soft number of blocking sequence can mating consensus sequence; With 3) consensus sequence is greater than 30 bases longs; S43, detect known fusion form: the comparison result analyzing S42, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And detect new fusion form: the comparison result analyzing S42, if not there is the record merged with first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.Wherein, namely the low-quality sequence pointed out in S4 does not meet above-mentioned 3 sequences required.
Apply technical scheme of the present invention, combining target areas captured technology, high throughput sequencing technologies and fusion sequence information analysis, provide the flow process of a set of highly sensitive detection fusion from DNA.Wherein, by the gene of catching normal generation fusion of target area capture technique, only the normal gene occurring to merge is checked order, greatly reduce order-checking cost; In addition, utilizing target area to catch lower fusion drives the single-ended fusion signal of gene to add that database can detect very high sensitivity to the fusion of the gene data that target area is caught.
Comparison in the present invention, adoptable software is as bwa-aln, bowtie2, clcgenomicsworkbench, according to a kind of typical embodiment of the present invention, what the comparison software in S41 adopted is BWA-mem comparison software, and the software setting up the employing of comparison result indexed file is samtools software.
According to a kind of typical embodiment of the present invention, use picard software in S42 removes the tumor-necrosis factor glycoproteins that PCR produces, many sequence is 4 sequences, and in S43, the aligned sequences that described homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.
According to a kind of typical embodiment of the present invention, provide a kind of device detecting gene fusion.This device comprises: DNA processing module, for extracting the DNA of sample to be detected, then interrupts, jointing; Gene trap module, the driving gene in the fusion to be checked of the method target acquisition gene utilizing target area to catch for using the Agilent probe customized according to target gene and then obtain target DNA fragments; Sequencer module, checks order to target DNA fragments for the method by high-flux sequence, obtains the sequence of fusion form; Detection module, after filtering out low-quality sequence, the sequence of detection fusion form; Detection module specifically comprises: sequence alignment submodule, the sequence of the fusion form obtained by detection module for utilizing comparison software and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sort according to the position of comparison, then set up the indexed file of comparison result; Sequence screening submodule, for the comparison result of the sequence merging form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing soft sequence of blocking sequence, the soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, require to include 1) block sequence at least 4 containing non-equal is soft, and can consensus sequence be mated; 2) the soft number of blocking sequence cannot mating consensus sequence contained is less than the soft number of blocking sequence can mating consensus sequence; With 3) consensus sequence is greater than 30nt; Detect known fusion form submodule: for the comparison result of analytical sequence screening submodule, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And detect new fusion form submodule: for the comparison result of analytical sequence screening submodule, if not there is the record merged with first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.
Preferably, what the comparison software in sequence screening submodule adopted is BWA-mem comparison software, and the software setting up the employing of comparison result indexed file is samtools software.
Preferably, the use picard software in sequence screening submodule removes the tumor-necrosis factor glycoproteins that PCR produces.
Preferably, many sequences in sequence screening submodule are 4 sequences.
Preferably, detect in new fusion form submodule, the aligned sequences that homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.Beneficial effect of the present invention is further illustrated below in conjunction with embodiment.
With reference to figure 1, following embodiment key step is as follows:
S1, extracts the DNA of sample to be detected, then interrupts, jointing;
S2, the driving gene in the fusion to be checked of the method target acquisition gene using the Agilent probe customized according to target gene to utilize target area to catch and then obtain target DNA fragments;
S3, is checked order to target DNA fragments by the method for high-flux sequence, obtains the sequence of fusion form;
S4, after filtering out low-quality sequence, the sequence of detection fusion form;
S4 specifically comprises:
S41, the sequence of described fusion form utilizing the just described S3 of BWA-mem comparison software to obtain and the reference sequences of corresponding fusion form compare, sequence in non-comparison forms soft blocking, then sort according to the position of comparison, and set up indexed file (comparison file) with samtools software;
S42, the comparison result of the sequence of described fusion form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing described soft sequence of blocking sequence, the described soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to described consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, described in require to include 1) block sequence at least 4 containing non-equal is soft, and described consensus sequence can be mated; 2) the soft number of blocking sequence cannot mating described consensus sequence contained is less than the soft number of blocking sequence can mating described consensus sequence; With 3) described consensus sequence is greater than 30nt;
S43, detect known fusion form: analyze the comparison result in all S42, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so regardless of comparison quality (whether aligned sequences has homologous sequence), all put in the result, and mark; And
Detect new fusion form: analyze the comparison result in all S42, if not there is the record merged in a database with first breakpoint in the region in comparison, so see comparison quality (whether aligned sequences has homologous sequence), remove low comparison quality, namely use comparison quality in BWA-mem default parameters comparison result to be less than 10 remove, will be left result output.
This software can detect the fusion sudden change of two types: a kind ofly to provide in COSMIC database, known common fusion, this fusion can find two merge breakpoints may region.Whether uniquely another the unknown do not had in a database newly merges form, and its breakpoint of this fusion does not have FX, but by, comparison can judge the confidence level of fusion with reference to genome.
Fig. 2 show assemble according to an embodiment of the invention soft block the step of sequence with judge the schematic diagram of confidence level.Fig. 2 is upper, and Gene A is the driving gene in merging, and gene B is chaperone (partnergene).In comparison, 5 sections of reading of A gene all start in this site to have and softly block sequence, front 4 articles of soft sequences of blocking can be mated completely with the part of the 3rd article, article 3, as consensus sequence, namely to require in 1 that non-equal is soft and block sequence at least 4, and can consensus sequence be mated.Article 5, soft sequence of blocking can not mate consensus sequence, namely requires that the soft number of blocking sequence cannot mating consensus sequence in 2 is less than can mate soft and blocks sequence number.In addition as requested 3 consensus sequences namely the 3rd article of soft length of blocking sequence must be greater than 30nt.In under Fig. 2, be the result of second time bwa-mem comparison, gene B and gene C are 2 chaperones to be selected, and Article 1 consensus sequence is can only uniquely in comparison on gene B, so Output rusults.Article 2 consensus sequence can gene B and gene C in comparison, if wherein have and drive gene fusion at the record of COSMIC lane database, so just marks and exports.
Embodiment 1
Sample to be detected is Lc-2/adRET fused cell system.In this following embodiment, build storehouse and use kapa reagent, capture agent is Agilent sure-select, if the step whether described in detail in embodiment, then the means of this area routine all can be adopted to realize.
1) extract sample DNA, utilize fluorescent quantitation meter (Qubit) to carry out quantitatively, its concentration is 3.8ng/ul, and volume is 130ul; Utilize Ultrasonic Cell Disruptor (Covaris) to carry out fragmentation to sample, make DNA fragmentation size between 200 ~ 400bp, then utilize agarose gel electrophoresis to detect clip size and whether meet the requirements.
2) first the sample of fragmentation is carried out magnetic beads for purifying, then carry out end reparation and 3 ' and hold polyadenylation, system configurations is in table 1, and basic step is as follows: first at 20 DEG C of temperature bath 30min, then terminates reaction at 65 DEG C of temperature bath 30min.
Table 1
| Polyadenylation damping fluid is held in end reparation and 3 ' | 7μl |
| End is repaired and 3 ' end adenylase mixed solution | 3μl |
| DNA | 50ul(500ng) |
3) DNA after above-mentioned reparation is carried out joint connection, connector interfaces is in table 2.Joint connects for bathing 15min 20 DEG C of temperature.
Table 2
Joint sequence is SEQIDNO:1GATCGGAAGAGCACACGTCTGAACTCCAGTCACTTAGGCATCTCGTA TGCCGTCTTCTGCTTG, and SEQIDNO:2AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTC GCCGTATCATT.
4) product after being connected by above-mentioned joint carries out magnetic beads for purifying, then carries out pcr amplification, obtains the DNA fragmentation of enough belt lacings, basic step is as follows: first at 98 DEG C of denaturation 45s, secondly at 98 DEG C of sex change 15s, then at 60 DEG C of annealing 30s, 72 DEG C extend 30s; Repeat sex change annealing extension process 7 times; Finally extend 1min at 72 DEG C, terminate reaction.Amplification system is in table 3.
Table 3
| Reagent | Volume |
| Rapid hot start polysaccharase | 25μL |
| Amplimer | 1uL |
| Connect the DNA fragmentation of joint | 24μL |
5) after magnetic beads for purifying being carried out to pcr amplification product, after utilizing Qubit quantitatively to obtain concentration, take out 500ng amplified production, use concentrating instrument by amplified production volume concentration to 4.4ul, then carry out closing and probe hybridization, hybridization system is as shown in table 4 below.
Table 4
| Reagent | Volume |
| Closed reagent mixed solution | 5.6μl |
| P5, P7 closed reagent | 2ul |
| Quick closure reagent | 5ul 6 --> |
| RNA enzyme closed reagent | 2ul |
| For the biotinylated probes of target area | 2ul |
| Hybridization buffer | 6ul |
| The water of nuclease free | 3ul |
| Pcr amplification product | 4.4ul |
Capture probe uses Agilent customized probe target acquisition gene, and probe covers all exons of RET gene and intron 7,9,10,11 region.
Hybridization condition is as shown in table 5 below.
Table 5
6) strepto-affinity biscuit porcelain pearl is used to catch the sample that probe combines, step is as follows: 50ul magnetic bead is added 1.5ml centrifuge tube, be placed on magnetic frame, abandon supernatant, after connecting buffer solution for cleaning three times with 200ul, 200ul is used to connect the resuspended magnetic bead of damping fluid, sample with probe hybridization is added magnetic bead, blending instrument is put upside down mixing 30min, be placed on magnetic frame, abandon supernatant, clean 1 time with scavenging solution 1, then clean 3 times with the scavenging solution 2 being preheating to 65 DEG C, period ensures that the temperature of magnetic bead and damping fluid 2 is at 65 DEG C.Finally be placed on magnetic frame, abandon supernatant, add the water of 38ul nuclease free, resuspended magnetic bead.
7) by magnetic capture to DNA fragmentation carry out pcr amplification, amplification system sees the following form 6, obtains enough DNA fragmentations adding top connection, basic step is as follows: first at 98 DEG C of denaturation 2min, secondly at 98 DEG C of sex change 30s, then at 60 DEG C of annealing 30s, 72 DEG C extend 1min; Repeat sex change annealing extension process 14 times; Finally extend 5min at 72 DEG C, terminate reaction.
Table 6
| Reagent | Volume |
| High-fidelity DNA polymerase | 1ul |
| Amplimer | 1ul |
| The mixed liquid of high-fidelity DNA polymerase reaction | 10ul |
| Mononucleotide mixed solution | 0.5ul |
| Target area domain dna on magnetic bead | 37.5ul |
8) pcr amplification product obtained is carried out magnetic beads for purifying, then utilize qPCR quantitative, utilize angilent2100bioanalyzer2100 to carry out clip size detection.
9) check order, NextSeq500 gene sequencer completes order-checking, it is that fq file stores all sequenced fragments results that the optical signal obtained is converted into machine data under base sequence by order-checking platform.
10) by reference genome in the comparison of lower machine data fq file, remove inferior quality sequence, the average order-checking degree of depth is 1175.77.
The sequence of detection fusion form, concrete steps refer to down: first have the soft sequential extraction procedures blocked out by all, then as Fig. 2 operation.Fig. 2 is upper, and Gene A is the driving gene in merging, and gene B is chaperone (partnergene).In comparison, 5 reads of A gene start in this site to have and softly block sequence, front 4 articles of soft sequences of blocking can be mated completely with the part of the 3rd article, article 3, as consensus sequence, namely to require in 1 that non-equal is soft and block sequence at least 4, and can consensus sequence be mated.Article 5, soft sequence of blocking can not mate consensus sequence, namely requires that the soft number of blocking sequence cannot mating consensus sequence in 2 is less than can mate soft and blocks sequence number.In addition as requested 3 consensus sequences namely the 3rd article of soft length of blocking sequence must be greater than 30nt.In under Fig. 2, be the result of second time bwa-mem comparison, gene B and gene C are 2 chaperones to be selected, and Article 1 consensus sequence is can only uniquely in comparison on gene B, so Output rusults.Article 2 consensus sequence can gene B and gene C in comparison, if wherein have and drive gene fusion at the record of COSMIC lane database, so just marks and exports.
Lc-2/adRET fused cell system check result is in table 7.
Table 7
| Sample number | RET holds total coverage | Merge check result |
| Lc-2/ad | 1222 | RET:exom11_CCDC6:intron_e1e2＝347 |
As can be seen from the above description, the above embodiments of the present invention achieve following technique effect:
First the mode using target area to catch in the present embodiment example, compared to genome sequencing in saving sequencing data amount, increase the order-checking degree of depth while the cost that namely checks order reduces can detect more low levels sudden change object to reach from the 9G of normal 30X genome sequencing to the 4G embodiment.If be used in the software of detection fusion in full-length genome in addition as CREST, result only has RET to have minority is longer softly blocks CCDC6 on sequence alignment, and owing to not catching, CCDC6 there is no soft sequence of blocking, cause finally normally detecting this and merge.And use above-mentioned fusion detection step and parameter accurately can detect this fusion.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. detect a method for gene fusion, it is characterized in that, comprise the following steps:
S1, extracts the DNA of sample to be detected, then interrupts, jointing;
S2, the method using the Agilent probe customized according to target gene to utilize target area to catch is caught the driving gene in the fusion to be checked of described target gene and then obtains target DNA fragments;
S3, is checked order to described target DNA fragments by the method for high-flux sequence, obtains the sequence of fusion form;
S4, after filtering out low-quality sequence, detects the sequence of described fusion form;
Described S4 specifically comprises:
S41, the sequence of described fusion form utilizing comparison software to be obtained by described S3 and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sorts according to the position of comparison, then sets up the indexed file of comparison result;
S42, the comparison result of the sequence of described fusion form is extracted, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing described soft sequence of blocking sequence, the described soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to described consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, described in require to include 1) block sequence at least 4 containing non-equal is soft, and described consensus sequence can be mated; 2) the soft number of blocking sequence cannot mating described consensus sequence contained is less than the soft number of blocking sequence can mating described consensus sequence; With 3) described consensus sequence is greater than 30nt;
S43, detect known fusion form: the comparison result analyzing described S42, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And
Detect new fusion form: the comparison result analyzing described S42, if not there is the record merged with described first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.
2. method according to claim 1, is characterized in that, what the comparison software in described S41 adopted is BWA-mem comparison software, and the software setting up the employing of described comparison result indexed file is samtools software.
3. method according to claim 1, is characterized in that, the use picard software in described S42 removes the tumor-necrosis factor glycoproteins that PCR produces.
4. method according to claim 1, is characterized in that, described many sequences in described S42 are 4 sequences.
5. method according to claim 1, is characterized in that, in described S43, the aligned sequences that described homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.
6. detect a device for gene fusion, it is characterized in that, comprising:
DNA processing module, for extracting the DNA of sample to be detected, then interrupts, jointing;
Gene trap module, catches the driving gene in the fusion to be checked of described target gene for the method using the Agilent probe customized according to target gene to utilize target area to catch and then obtains target DNA fragments;
Sequencer module, checks order to described target DNA fragments for the method by high-flux sequence, obtains the sequence of fusion form;
Detection module, after filtering out low-quality sequence, detects the sequence of described fusion form;
Described detection module specifically comprises:
Sequence alignment submodule, the sequence of described fusion form obtained by described detection module for utilizing comparison software and the reference sequences of corresponding fusion form compare, sequence in non-comparison is formed softly blocks sequence, and sort according to the position of comparison, then set up the indexed file of comparison result;
Sequence screening submodule, comparison result for the sequence by described fusion form extracts, remove the tumor-necrosis factor glycoproteins that PCR produces, find containing described soft sequence of blocking sequence, the described soft sequence of blocking of many sequence supports of same point is assembled, obtain consensus sequence, filter out low-quality sequence according to described consensus sequence, remaining satisfactory sequence is continued comparison; Wherein, described in require to include 1) block sequence at least 4 containing non-equal is soft, and described consensus sequence can be mated; 2) the soft number of blocking sequence cannot mating described consensus sequence contained is less than the soft number of blocking sequence can mating described consensus sequence; With 3) described consensus sequence is greater than 30nt;
Detect known fusion form submodule: for analyzing the comparison result of described sequence screening submodule, if comparison has the record occurring with first breakpoint to merge in COSMIC database to the region on reference sequences, so no matter whether aligned sequences has homologous sequence, all put in the result, and mark; And
Detect new fusion form submodule: for analyzing the comparison result of described sequence screening submodule, if not there is the record merged with described first breakpoint in the region in comparison in COSMIC database, so see whether aligned sequences has homologous sequence, remove the aligned sequences that homology is low, result will be left and export.
7. device according to claim 6, is characterized in that, what the comparison software in described sequence screening submodule adopted is BWA-mem comparison software, and the software setting up the employing of described comparison result indexed file is samtools software.
8. device according to claim 6, is characterized in that, the use picard software in described sequence screening submodule removes the tumor-necrosis factor glycoproteins that PCR produces.
9. device according to claim 6, is characterized in that, described many sequences in described sequence screening submodule are 4 sequences.
10. device according to claim 6, is characterized in that, in the fusion form submodule that described detection is new, the aligned sequences that described homology is low refers to and uses the sequence that in BWA-mem default parameters comparison result, comparison quality is less than 10.
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