CN106815491B - Device for detecting gene fusion of FFPE sample - Google Patents
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Abstract
The invention relates to a device for detecting gene fusion of an FFPE (fringe field programmable gate array) sample, which comprises a sequencing data acquisition module, a comparison module, a re-comparison module, a real fusion breakpoint judgment module and an output module. The device for detecting the gene fusion of the FFPE sample has the advantages of high detection speed, low resource requirement and high stability.
Description
Technical Field
The invention relates to the field of gene fusion detection, in particular to a device and a method for detecting FFPE sample gene fusion.
Background
Tissue specimens prepared by Formalin-fixed Paraffin-embedded (FFPE) methods are called Formalin-fixed Paraffin-embedded tissue samples, FFPE samples for short. The FFPE sample can be preserved for a long time, is commonly used for clinical pathological examination, tumor gene detection and medical science research, and is a reliable material source for molecular biological research.
Worldwide, there are approximately billions of tissue samples kept in hospitals or tissue sample banks. The vast majority of which are FFPE samples. FFPE samples generally represent valuable and widely available biomedical research materials, and a huge number of archived FFPE samples provide valuable resources for retrospective studies, elucidation of disease mechanisms, discovery of therapeutic targets, and indication of prognosis. In particular, there are a large number of tumor tissue sections that are stored in the form of FFPE samples.
Fusion genes are a kind of chromosome structural variation which is very important in clinic and play a key role in the process of cancer occurrence and development. The accurate detection result of the fusion gene can provide reference basis for clinical anticancer target medication treatment and prognosis evaluation.
Detection techniques traditionally used to detect fusion genes are based primarily on genetic methods, such as FISH. However, the relatively low resolution and throughput limits the application of this method to the detection of complex epithelial cancers.
With the development of the second generation sequencing technology, a large number of detection methods for detecting fusion genes emerge. In the gene fusion detection method, the determination of the detection result is directly influenced by the confirmation of the breakpoint. CREST is one of the mainstream algorithms for detecting Fusion gene at present, and the algorithm utilizes an assembly algorithm to realize two-time assembly so as to eliminate false positives, so that the method has the main advantage of low false positives, but has the defects of low detection speed, high resource requirement, assembly requirement and the like due to the fact that two-time assembly is required; at the same time, the assembly effect is also influenced by the coverage and the insert length. Due to the high degradation factor of the FFPE sample, the fragment length is short, the coverage is reduced, and the length of the assembled fragment is greatly influenced, so that the fusion detection result is influenced. Therefore, how to detect the fusion gene of the FFPE sample becomes an urgent problem in the art.
Disclosure of Invention
Technical problem to be solved by the invention
The algorithm in the prior art needs to perform two times of assembly and three times of comparison, so that the defects of low detection speed, high resource requirement and the like exist, and meanwhile, because the assembly sequences of the FFPE samples are short and low in coverage, certain uncertainty exists in the assembly of repeated sequences, and the detection result can be wrong.
In view of the problems of the prior art, the present invention is directed to an apparatus and a method for detecting gene fusion, which have the advantages of high detection speed, low resource requirement and high stability.
Compared with the prior art, the detection device provided by the invention fully utilizes the information of the sequencing fragments (reads) of the PE sequencing off-machine, reduces the comparison times, only needs two comparisons, does not need to be assembled, and improves the detection stability.
Namely, the present invention comprises:
an apparatus for detecting FFPE sample gene fusion, comprising the following modules:
the sequencing data acquisition module is used for acquiring the sequencing data of the FFPE sample; preferably, the Sequencing data is obtained by using a Paired-end Sequencing (PE Sequencing) method;
A comparison module: the sequencing data acquisition module is connected with the sequencing data acquisition module and is used for comparing the acquired sequencing data with a reference sequence to acquire a comparison result. The alignment result comprises the corresponding position information of the sequencing fragment in the gene. The position information comprises soft cutting information and successful comparison information. And the part with the soft shearing information in the sequencing fragment is a soft shearing part of the sequencing fragment, and the part with the successful comparison information in the sequencing fragment is a successful comparison part of the sequencing fragment. Preferably, the module can use bwa software to find the corresponding position of the sequencing fragment in the gene and form a bam format file; preferably, the bam file comprises description information (qname), sequence information (seq), alignment Position (POS), bit identifier (flag), alignment quality value (MAPQ), brief alignment expression information (Cigar), template length (Tlen) of each sequencing fragment;
and a re-comparison module: the sequencing fragment comparison module is connected with the comparison module and is used for comparing the sequencing fragment with the soft shearing information with the reference genome again to obtain a re-comparison result;
real integration breakpoint judgment module: the re-comparison module is connected with the sequencing fragment and is used for judging the fusion breakpoint of the sequencing fragment; and
An output module: it is connected to the real fusion breakpoint determination module and is used for outputting gene fusion detection results, for example, gene fusion breakpoint positions (e.g., left _ pos, right _ pos), chromosome numbers (e.g., left _ chr, right _ chr), support degrees (e.g., sup), and the like.
Preferably, the realignment module may, for example, comprise the following sub-modules:
length filter submodule: the sequencing module is connected with the comparison module and is used for filtering and removing sequencing fragments with the length smaller than a certain value in the sequencing fragments containing soft-clipping information; preferably, the certain value may be, for example, 15 to 30bp, preferably 20 to 25 bp.
A breakpoint judgment submodule: the length filtering submodule is connected with the length filtering submodule and is used for taking the joint of the part with the soft shearing information and the part with the normal comparison information in the sequencing fragment as a breakpoint according to the result data of the length filtering submodule;
distinguishing sub-modules: the breakpoint judgment sub-module is connected with the soft cutting information part and the normal comparison information part, and is used for separating the soft cutting information part and the normal comparison information part at a breakpoint and respectively storing sequence information of the two parts into two files (such as fastq files);
And then comparing the submodules: the distinguishing submodule is connected with the two files respectively storing the sequence information and the reference sequence and is used for comparing the two files respectively storing the sequence information with the reference sequence again to obtain a re-comparison result; preferably, the realignment result includes the following information: description information (qname), sequence information (seq), alignment Position (POS), bit identifier (flag), alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment. Preferably, the above two fastq files are compared again by using bwa software, for example, to form a bam format file. The bam format file comprises description information (qname), sequence information (seq), a bit identifier (flag), an alignment Position (POS), an alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment.
Preferably, the real fusion breakpoint judgment module may include the following sub-modules: a filtering submodule: the re-comparison submodule is connected with the re-comparison submodule and is used for filtering and removing sequencing fragments which are not successfully compared (mapped) and sequencing fragments with low contrast mass value (MAPQ) according to a bit identifier (flag) value;
a breakpoint information acquisition submodule: the sequencing sub-module is connected with the filtering sub-module and is used for searching sequencing fragments with the same fragment description information (qname) and acquiring breakpoint information; preferably, the breakpoint information includes: (1) left/right _ chr, chromosome number of breakpoint Left/right sequences; (2) left/right _ pos, alignment position of the first base left/right of the breakpoint; (3) left/right _ seq, sequence of base left/right of breakpoint; (4) sup, breakpoint support, number of sequencing fragments supporting the breakpoint.
Fusing breakpoint screening submodules: the breakpoint information acquisition submodule is connected with the breakpoint information acquisition submodule and is used for screening fusion breakpoints in breakpoint information;
merging the break points and merging the sub-modules for the first time: the fusion breakpoint screening submodule is connected with the fusion breakpoint screening submodule and is used for merging the fusion breakpoints with the same breakpoint information into a real fusion breakpoint, and taking the number of the fusion breakpoints with the same breakpoint information as the support degree of the real fusion breakpoint. The same breakpoint information means that left _ chr, left _ pos, right _ chr and right _ pos are the same.
Merging the sub-modules again by fusing breakpoints: and the break point primary merging submodule is connected with the break point primary merging submodule and is used for merging the fusion break points which are the same as left _ chr and right _ chr and have a difference of a certain value (for example, 3bp) between right _ pos and left _ pos into a real fusion break point.
Preferably, the breakpoint information includes:
left _ chr: chromosome number of the sequence to the left of the breakpoint, reference sequence number corresponding to read 1.
left _ pos alignment position of the first base at the left of the breakpoint, alignment position corresponding to read1 plus sequence length of read 1.
left _ seq: the sequence of bases to the left of the breakpoint.
right _ chr: chromosome number of the right sequence of the breakpoint, reference sequence number corresponding to read 2.
right _ pos: the alignment position of the first base to the right of the breakpoint, the alignment position corresponding to read2 plus the sequence length of read 2.
right _ seq: the sequence of bases to the right of the breakpoint.
sup: and the breakpoint support degree, namely the number of sequencing fragments supporting the breakpoint, is 1 by default.
In addition, the breakpoint information may also include, ort: judging according to the comparison result mode in the fragment description information in the sequencing fragment, wherein "+" represents that soft shearing occurs on the right side of the break point of the clean sequencing fragment, and "-" represents that soft shearing occurs on the left side of the break point of the clean sequencing fragment.
Preferably, the fusion breakpoint screening submodule includes the following elements:
breakpoint quality filter element: for filtering low quality breakpoints, if there is a breakpoint a, the sup number in a is greater than a certain value (e.g. 5), the comparison quality values in left _ seq and right _ seq are both greater than a certain value (e.g. 30), the mismatch rates are both less than a certain value (e.g. 0.05) or/and the breakpoint support/breakpoint right or left position depth is greater than a certain value (e.g. 0.1), then the breakpoint a is a fusion breakpoint.
Same breakpoint merge element: merging the breakpoints A and B into a merged breakpoint if the breakpoints A and B exist, left _ chr in A is equal to right _ chr in B, right _ chr in A is equal to left _ chr in B, left _ pos in A is equal to right _ pos in B, and right _ pos in A is equal to left _ pos in B;
Preferably, according to the above-mentioned information of fusion breakpoints, if there are a fusion breakpoint a where right _ pos and a fusion breakpoint B where right _ pos are smaller than a certain value (e.g., 5), and a fusion breakpoint a where left _ pos and a fusion breakpoint B where left _ pos are smaller than a certain value (e.g., 5), the fusion breakpoint a and the fusion breakpoint B are merged into a real fusion breakpoint. Thereby finally obtaining the detection result of gene fusion (gene fusion).
In addition, the present invention further comprises:
a method for detecting FFPE sample gene fusion, comprising the steps of:
a sequencing data acquisition step, namely acquiring the sequencing data of the FFPE sample; preferably, the Sequencing data is obtained by using a Paired-end Sequencing (PE Sequencing) method;
and (3) comparison: and comparing the obtained sequencing data with the reference sequence to obtain a comparison result. The alignment result comprises the corresponding position information of the sequencing fragment in the gene. The position information comprises soft cutting information and successful comparison information. And the part with the soft shearing information in the sequencing fragment is a soft shearing part of the sequencing fragment, and the part with the successful comparison information in the sequencing fragment is a successful comparison part of the sequencing fragment. Preferably, the module can use bwa software to find the corresponding position of the sequencing fragment in the gene and form a bam format file; preferably, the bam file comprises description information (qname), sequence information (seq), alignment Position (POS), bit identifier (flag), alignment quality value (MAPQ), brief alignment expression information (Cigar), template length (Tlen) of each sequencing fragment;
And (3) comparing: comparing the sequencing fragment with the soft shearing information with the reference genome again to obtain a re-comparison result;
and a step of judging true fusion breakpoints: judging the fusion breakpoint of the sequencing fragment; and
an output step: and outputting the gene fusion detection result, such as breakpoint position (e.g. left _ pos, right _ pos), chromosome number (e.g. left _ chr, right _ chr), support degree (e.g. sup) and the like.
Preferably, the realignment step may, for example, comprise the following sub-steps:
a length filtering substep: filtering to remove sequencing fragments with the length smaller than a certain value in the sequencing fragments containing soft-clipping (soft-clipping) information; preferably, the certain value may be, for example, 15 to 30bp, preferably 20 to 25 bp.
And a breakpoint judgment substep: according to the result data of the length filtering submodule, taking the joint of the part with the soft shearing information and the part with the normal comparison information in the sequencing fragment as a breakpoint;
a distinguishing substep: separating the part with the soft cutting information and the part with the normal comparison information at a break point, and respectively storing the sequence information of the two parts into two files (such as fastq files);
And a comparison substep: comparing the two files respectively storing the sequence information with the reference sequence again to obtain a re-comparison result; preferably, the realignment result includes the following information: description information (qname), sequence information (seq), alignment Position (POS), bit identifier (flag), alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment. Preferably, the above two fastq files are compared again by using bwa software, for example, to form a bam format file. The bam format file comprises description information (qname), sequence information (seq), a bit identifier (flag), an alignment Position (POS), an alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment.
Preferably, the step of determining the true fusion breakpoint may include the following sub-steps: a filtering substep: filtering and removing sequencing fragments which are not successfully aligned (unmapped) and sequencing fragments with low ratio-to-quality value (MAPQ) according to a bit identifier (flag) value;
breakpoint information acquisition substep: searching sequencing fragments with the same fragment description information (qname), and acquiring breakpoint information; preferably, the breakpoint information includes: (1) left/right _ chr, chromosome number of breakpoint Left/right sequences; (2) left/right _ pos, alignment position of the first base left/right of the breakpoint; (3) left/right _ seq, sequence of base left/right of breakpoint; (4) sup, breakpoint support, number of sequencing fragments supporting the breakpoint.
And a fusion breakpoint screening substep: screening fusion breakpoints from the breakpoint information;
and a fused breakpoint primary merging sub-step: merging the fusion breakpoints with the same breakpoint information into a real fusion breakpoint, and taking the number of the fusion breakpoints with the same breakpoint information as the support degree of the real fusion breakpoint. The same breakpoint information means that left _ chr, left _ pos, right _ chr and right _ pos are the same.
Merging the merged breakpoints again: the fusion break points with left _ chr and right _ chr being the same and right _ pos or left _ pos within a certain value (for example, 3bp) are combined into a real fusion break point.
Preferably, the breakpoint information includes:
left _ chr: chromosome number of the sequence to the left of the breakpoint, reference sequence number corresponding to read 1.
left _ pos alignment position of the first base at the left of the breakpoint, alignment position corresponding to read1 plus sequence length of read 1.
left _ seq: the sequence of bases to the left of the breakpoint.
right _ chr: chromosome number of the right sequence of the breakpoint, reference sequence number corresponding to read 2.
right _ pos: the alignment position of the first base to the right of the breakpoint, the alignment position corresponding to read2 plus the sequence length of read 2.
right _ seq: the sequence of bases to the right of the breakpoint.
sup: and the breakpoint support degree, namely the number of sequencing fragments supporting the breakpoint, is 1 by default.
Preferably, the breakpoint screening submodule includes the following steps:
if there is a break point a, the sup number in a is greater than a certain value (e.g. 5), the comparison quality values in left _ seq and right _ seq are both greater than a certain value (e.g. 30), the mismatch rates are both less than a certain value (e.g. 0.05) or/and the break point support/break point right or left position depth is greater than a certain value (e.g. 0.1), then the break point a is determined to be a fusion break point.
If the breakpoints A and B exist, left _ chr in A is equal to right _ chr in B, right _ chr in A is equal to left _ chr in B, left _ pos in A is equal to right _ pos in B, and right _ pos in A is equal to left _ pos in B, the breakpoints A and B are merged into a merged breakpoint;
preferably, according to the above fusion breakpoint information, if there are fusion breakpoints a and B in which right _ pos is smaller than a certain value (e.g., 5) and fusion breakpoints a and B in which left _ pos is smaller than a certain value (e.g., 5), the fusion breakpoints a and B are merged into a real fusion breakpoint, so as to finally obtain a gene fusion (gene fusion) detection result.
According to the invention, the device and the method for detecting the gene fusion of the FFPE sample are high in detection speed, low in resource requirement and high in stability. In the second and third comparison processes of the existing algorithm, only one sequence is compared each time, and system resources are occupied for a long time. Compared with the existing algorithm, the generation algorithm of the invention fully utilizes the advantages of PE sequencing, reduces the comparison times and only adopts twice comparison. Filtering to obtain all fragments (sequencing fragments containing soft-cutting information) which are possible to be fused during the first alignment; the second comparison is to compare all the sequences at the same time, so that the utilization rate of system resources is improved. In addition, the algorithm does not need to assemble the sequence, and has no instability caused by assembly, thereby realizing the gene fusion detection of the FFPE sample.
Drawings
FIG. 1 is a schematic view of the apparatus for detecting gene fusion of an FFPE sample according to example 1.
FIG. 2 is a schematic view showing an example of a conventional apparatus for detecting gene fusion.
Detailed description of the invention
Technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art, and in case of conflict, the definitions in this specification shall control.
In general, the terms used in the present specification have the following meanings.
Reference sequence (Refseq) species reference standard genomic sequence.
Fusion gene (Fusion gene) refers to a process in which sequences of all or a part of two genes are fused to each other to form a new gene. It may be the result of a chromosomal translocation, an intermediate deletion or a chromosomal event.
Reads genomic or transcriptome sequence fragments.
PE sequencing: paired-end sequencing, a sequencing method.
read 1/2: in the PE sequencing data, read1 represents the nucleotide sequence obtained in the first round of the test, and read2 represents the nucleotide sequence obtained in the second round of the test.
bwa: a comparison method software is used for searching the position of reads in Refseq, and finally obtaining a bam format file.
adapter sequence adapter sequences flanking the DNA fragment in the sequencing.
Breakpoint (breakpoint): the point at which the two gene sequences in the fusion gene are linked to each other.
soft-clipping reads: after the reads are compared, if part of the sequences are compared to a certain position of the Refseq and the other part of the sequences are compared to the other position of the Refseq or cannot be compared to the Refseq, the reads are called soft-clipping reads.
flag: one value used for describing information such as sequence alignment mode, direction and the like in the bam format file
cigar: a brief alignment information expression, which represents the alignment results using data plus letters based on the reference sequence.
unmapped reads means that reads is not aligned to a position in the Refseq.
duplicate: repetitive sequence refers to a sequence amplified by PCR.
Fragment description information: qname, descriptor of alignment fragment (template).
In the comparison process, certain difference between reads and Refseq can be allowed, and the ratio of the difference value to the length of the reads is compared with the mismatch rate.
Comparing the quality values: indicating the likelihood of aligning to the wrong location, with higher values indicating lower likelihood.
Examples
The present invention will be described more specifically with reference to the following examples, but the present invention is not limited to these examples.
Example 1 apparatus for detecting FFPE sample gene fusion according to the present invention
The device for detecting the gene fusion of the FFPE sample is used for detecting the gene fusion condition of the tissue FFPE sample of a female lung cancer patient.
The FFPE sample copy number variation detection device is used for detecting the gene fusion condition of a tissue FFPE sample of a female patient with lung adenocarcinoma.
1.1 extraction of DNA from FFPE samples
The FFPE sample DNA was obtained by performing extraction procedures using the GeneRead DNA FFPE Kit (QIAGEN Co.) according to the manual.
1.2 sample disruption
And (3) using a Biorupter interrupt instrument to perform interruption, setting interrupt conditions for 30 cycles, and setting 30s ON/30s OFF to interrupt the FFPE sample DNA into fragments of about 200bp to obtain the fragmented DNA fragments.
1.3 End Repair (End Repair)
(1) The required reagents were removed from the kit stored at-20 ℃ in advance and the individual sample amounts are shown in Table 1.
TABLE 1
(2) End repair reaction: after the addition of the DNA sample, the 1.5mL centrifuge tube was placed in a Thermomixer and incubated at 20 ℃ for 30 minutes. After completion of the reaction, DNA in the purification reaction system was collected using 1.8X nucleic acid purification beads and dissolved in 32. mu.LEB.
1.4 adding A (A-Tailing) at the end
(1) The required reagents were removed from the kit stored at-20 ℃ in advance, and the single sample formulation amounts are shown in table 2:
TABLE 2
(2) And (3) adding A at the tail end for reaction: after adding 32. mu.L of the DNA recovered by the previous purification step, a 1.5mL centrifuge tube was placed in a Thermomixer and incubated at 37 ℃ for 30 minutes. DNA in the purification reaction system was recovered using 1.8X nucleic acid purification magnetic beads and dissolved in 18. mu.L EB.
1.5 connection of the Joint (Adapter Ligation)
(1) The required reagents were removed from the kit stored at-20 ℃ in advance, and the single sample formulation amounts are shown in table 3:
TABLE 3
(2) And (3) connecting the joint: after adding 18. mu.L of the recovered DNA purified in the previous step, the sample tube was incubated in a Thermomixer at 20 ℃ for 15 minutes. DNA in the purification reaction system was recovered using 1.8 Xnucleic acid purification magnetic beads and dissolved in 30. mu.L of EB.
1.6PCR reaction
(1) Taking out the required reagent from the kit stored at the temperature of-20 ℃, and preparing a PCR reaction system in a 2mL PCR tube:
TABLE 4
(2) The PCR program was set, and the program for the PCR reaction was set as follows:
and (5) taking out the sample in time after the reaction is finished, storing the sample in a refrigerator at 4 ℃, and withdrawing or closing the instrument according to requirements.
(3) DNA in the purification reaction system was recovered using 0.9 Xnucleic acid purification magnetic beads, and the purified library was dissolved in 20. mu.L of ddH2And (4) in O. The library was subjected to the Qubit assay and submitted to the Agilent 2100.
1.7 Lung cancer target region Capture chip library hybridization
(1) In this experiment, buffers for providing an ionic environment for the hybridization capture reaction, and washing solutions and rinsing solutions for eluting physical adsorption or nonspecific hybridization were commercially available.
(2) Preparing a hybridization library: the DNA library to be hybridized is thawed on ice and 1. mu.g of total mass is taken (this DNA library is referred to as sample library in the subsequent working up step).
(3) Preparation of an Ann primer Pool: the tag primer In1 (100. mu.M) and the common primer (1000. mu.M) corresponding to the sample library Index were mixed together at 1000pmol each (this mixture was called Ann primer pool In the subsequent operation).
(4) Preparation of hybridization samples: to a 1.5mL EP tube was added 5. mu.L of COT DNA (Human COT-1DNA, Life technologies, 1mg/mL), 1. mu.g of the sample library, and an primer pool. The prepared hybridization sample EP tube was sealed with a sealing film, and the EP tube containing the sample library pool/COT DNA/Ann primer pool was placed in a vacuum apparatus until completely dried.
(5) Solution of hybridization sample: to a dry powder of sample library pool/COT DNA/Ann primer pool was added:
7.5 μ L of 2 × hybridization buffer
3 μ L of hybridization fraction A
(6) After mixing well, the mixture was denatured for 10 minutes on a pre-prepared 95 ℃ heating module.
(7) The mixture was transferred to a 0.2mL flat-capped PCR tube containing 4.5. mu.L of the capture chip. Vortex well for 3 seconds and place the hybridization sample mixture on a 47 ℃ heating block for 16 hours. The temperature of the heat cover of the heating module needs to be set to 57 ℃, and the product after hybridization needs to be subjected to subsequent elution and recovery operation.
(8) 10 Xthe cleaning solution (I, II and III), 10 Xthe rinsing solution and 2.5 Xthe magnetic bead cleaning solution were prepared into 1 Xthe working solution.
TABLE 5
(9) The following reagents were preheated in a 47 ℃ heating module:
400 μ L of 1 × rinsing liquid
100 μ L of 1 XWash solution I
1.8 preparation of affinity adsorption magnetic beads
(1) Streptavidin magnetic beads (Dynabeads M-280Streptavidin, hereinafter referred to as magnetic beads) were equilibrated at room temperature for 30 minutes, and then the beads were vortexed thoroughly for 15 seconds.
(2) 100 mu L of magnetic beads are subpackaged in a 1.5mL centrifuge tube, the centrifuge tube containing 100 mu L of magnetic beads is placed on a magnetic frame, after about 5 minutes, the supernatant is carefully discarded, 1 Xmagnetic bead cleaning solution with the volume twice that of the initial volume of the magnetic beads is added, and the mixture is vortexed and mixed for 10 seconds. And (4) putting the centrifugal tube containing the magnetic beads back to the magnetic frame to adsorb the magnetic beads. After the solution was clear, the supernatant was discarded by aspiration. The procedure was repeated twice for a total of two washes.
(3) After washing, the magnetic bead washing solution was aspirated, and the magnetic beads were resuspended in a 1 × magnetic bead washing solution of the initial volume of the magnetic beads by vortexing and transferred to a 0.2mL PCR tube. Placing the PCR tube on a magnetic frame to adsorb magnetic beads for clarification, and then absorbing and removing the supernatant.
1.9 binding and rinsing of DNA and affinity adsorption magnetic beads
(1) And transferring the hybridized sample library into a 0.2mL PCR tube containing affinity adsorption magnetic beads, and performing vortex oscillation and uniform mixing.
(2) The 0.2mL PCR tube was placed in a 47 ℃ heating block for 45 minutes and vortexed once every 15 minutes to bind the DNA to the beads.
(3) After 45 min incubation, 100. mu.L of 1 XWash solution I pre-warmed at 47 ℃ was added to 15. mu.L of the captured DNA sample. Vortex for 10 seconds. All the components in the 0.2mL PCR tube were transferred to a 1.5mL centrifuge tube. A1.5 mL centrifuge tube was placed on a magnetic rack to adsorb magnetic beads, and the supernatant was discarded.
(4) A1.5 mL centrifuge tube was removed from the magnetic rack and 200. mu.L of a preheated 47 ℃ 1 Xrinse was added. Sucking and mixing for 10 times (rapid operation is needed, and the temperature of the reagent and the sample is prevented from being lower than 47 ℃). After mixing, the sample was placed on a heating module at 47 ℃ for 5 minutes. This procedure was repeated and washed twice with 1 × rinse at 47 ℃. A1.5 mL centrifuge tube was placed on a magnetic rack, magnetic beads were adsorbed, and the supernatant was discarded.
(5) 200. mu.L of room temperature 1 XWash I was added to the 1.5mL centrifuge tube and vortexed for 2 minutes. Placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads, and discarding the supernatant. 200. mu.L of room temperature 1 XWash II was added to the 1.5mL centrifuge tube and vortexed for 1 minute. Placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads, and discarding the supernatant. 200. mu.L of room temperature 1 XWash III was added to the above 1.5mL centrifuge tube and vortexed for 30 seconds. Placing the centrifuge tube on a magnetic frame, adsorbing magnetic beads, and discarding the supernatant.
(6) The 1.5mL centrifuge tube was removed from the magnetic rack, and 45. mu.L of PCR water was added to dissolve the eluted magnetic beads to capture the sample.
1.10 PCR amplification of captured DNA
(1) The post-capture PCR mix was prepared according to the following table, and vortexed and mixed well after preparation. Both the enriching primer F and the enriching primer R were purchased from Yingchi Weiji Co.
(2) The amplification program of magnetic bead adsorption DNA PCR was set as follows:
(3) recovery and purification of hybridization capture DNA PCR product: the DNA in the purification reaction system was recovered using nucleic acid purification magnetic beads in an amount of 0.9X, and the purified library was dissolved in 30. mu.L of ddH2And (4) in O.
1.11 library quantitation
The library was subjected to 2100Bio Analyzer (Agilent)/LabChip GX (Caliper) and QPCR assays and the library concentration was recorded.
1.12 on-machine sequencing of libraries
The constructed library was sequenced using NextSeq 550AR (PE 100).
1.13 data processing and analysis
The device for detecting the gene fusion of the FFPE sample is adopted to process and analyze the result of the on-machine sequencing of the 1.12 library.
The device for detecting FFPE sample gene fusion comprises:
and the sequencing data acquisition module is used for acquiring lung cancer FFPE samples to be detected by using the lung cancer target area capture chip to capture and sequence so as to obtain sequencing data.
A comparison module: the sequencing data acquisition module is connected with the sequencing data acquisition module and is used for comparing the acquired sequencing data with a reference sequence to acquire a comparison result. The alignment results include the corresponding positions of the sequenced fragments in the reference sequence. The position information comprises soft cutting information and successful comparison information. And the part with the soft shearing information in the sequencing fragment is a soft shearing part of the sequencing fragment, and the part with the successful comparison information in the sequencing fragment is a successful comparison part of the sequencing fragment. The module searches the corresponding position of the sequencing fragment in the gene by using bwa software and forms a bam format file; the bam file comprises description information (qname), sequence information (seq), comparison Position (POS), bit identifier (flag), comparison quality value (MAPQ), brief comparison expression information (Cigar) and template length (Tlen) of each sequencing fragment.
And a re-comparison module: the system is connected with the comparison module and used for comparing the sequencing fragment with the soft shearing information with the reference genome again to obtain a re-comparison result.
The realignment module includes the following sub-modules:
length filter submodule: the sequencing fragment is connected with the alignment module and used for filtering and removing sequencing fragments with the length less than 20bp in the sequencing fragments containing soft-clipping (soft-clipping) information.
A breakpoint judgment submodule: the length filter submodule is connected with the length filter submodule and is used for taking the joint of the part with the soft shearing information and the part with the normal comparison information in the sequencing fragment as a breakpoint according to the result data of the length filter submodule.
Distinguishing sub-modules: the breakpoint judgment sub-module is connected with the soft cutting information part and the normal comparison information part, and is used for separating the soft cutting information part and the normal comparison information part at a breakpoint and respectively storing the sequence information of the two parts into two fastq files.
And then comparing the submodules: the distinguishing submodule is connected with the two files respectively storing the sequence information and the reference sequence and is used for comparing the two files respectively storing the sequence information with the reference sequence again to obtain a re-comparison result; the re-alignment results include: description information (qname), sequence information (seq), alignment Position (POS), bit identifier (flag), alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment. And comparing the two fastq files again by using bwa software to form a bam format file. The bam format file comprises description information (qname), sequence information (seq), a bit identifier (flag), an alignment Position (POS), an alignment quality value (MAPQ), brief alignment expression information (Cigar) and template length (Tlen) of each sequencing fragment.
Real integration breakpoint judgment module: the system is connected with the re-comparison module and is used for judging the fusion breakpoint of the sequencing fragment.
The real fusion breakpoint judgment module comprises the following sub-modules:
a filtering submodule: the re-comparison submodule is connected with the re-comparison submodule and is used for filtering and removing sequencing fragments which are not successfully compared (mapped) and sequencing fragments with low contrast mass value (MAPQ) according to a bit identifier (flag) value;
a breakpoint information acquisition submodule: the sequencing sub-module is connected with the filtering sub-module and used for searching sequencing fragments with the same fragment description information and acquiring breakpoint information. The breakpoint information includes: (1) left _ chr: chromosome number of the sequence to the left of the breakpoint, reference sequence number corresponding to read 1. (2) left _ pos alignment position of the first base at the left of the breakpoint, alignment position corresponding to read1 plus sequence length of read 1. (3) left _ seq: the sequence of bases to the left of the breakpoint. (4) right _ chr: chromosome number of the right sequence of the breakpoint, reference sequence number corresponding to read 2. (5) right _ pos: the alignment position of the first base to the right of the breakpoint, the alignment position corresponding to read2 plus the sequence length of read 2. (6) right _ seq: the sequence of bases to the right of the breakpoint. (7) sup: and the breakpoint support degree, the number of sequencing fragments supporting the breakpoint, is 1 by default.
Fusing breakpoint screening submodules: the breakpoint information acquisition submodule is connected with the breakpoint information acquisition submodule and is used for screening fusion breakpoints in breakpoint information.
The fusion breakpoint screening submodule comprises the following elements:
breakpoint quality filter element: for filtering out low quality breakpoints. If the breakpoint A exists, the sup number in the breakpoint A is more than 5, the comparison quality values in the left _ seq and the right _ seq are both more than 30, and the mismatching rates are both less than 0.05, the breakpoint A is judged to be the fusion breakpoint.
Same breakpoint merge element: for merging the same breakpoints. If the breakpoints A and B exist, left _ chr in A is equal to right _ chr in B, right _ chr in A is equal to left _ chr in B, left _ pos in A is equal to right _ pos in B, and right _ pos in A is equal to left _ pos in B. A and B are two forms of the same breakpoint, and the breakpoint A and the breakpoint B are combined into a fusion breakpoint.
Merging the break points and merging the sub-modules for the first time: the breakpoint combination module is connected with the fusion breakpoint screening submodule and used for combining breakpoints with the same breakpoint information (left _ chr, left _ pos, right _ chr and right _ pos are all the same) into a real fusion breakpoint, and taking the number of the breakpoints with the same breakpoint information as the support degree of the real fusion breakpoint.
Merging the sub-modules again by fusing breakpoints: and the merging sub-module is connected with the breakpoint primary merging sub-module, and merges the fusion breakpoints which are identical to left _ chr and right _ chr but have the difference of 5bp between right _ pos and left _ pos into a real fusion breakpoint. And the fusion breakpoint recombining module is used for merging the fusion breakpoint A and the fusion breakpoint B into a gene fusion breakpoint (gene fusion) if the right _ pos in the fusion breakpoint A and the right _ pos in the fusion breakpoint B are smaller than 5 and the left _ pos in the fusion breakpoint A and the left _ pos in the fusion breakpoint B are smaller than 5 according to the fusion breakpoint information. Thereby finally obtaining the gene fusion detection result. And
An output module: the real fusion breakpoint judgment module is connected with the real fusion breakpoint judgment module and is used for outputting a gene fusion detection result. The results of the measurements are shown in the following table.
1.14 validation of results
And (3) verifying the residual FFPE samples of the same patient by adopting a QPCR method, and detecting whether the EML4-ALK fusion occurs. Firstly, RNA extraction is carried out on the residual FFPE sample, and the specific steps refer to the steps of a Qiagen FFPERNA extraction kit (MagMAX)TMFFPE DNA/RNA Ultra Kit). The detection result shows that the EML4 is fused with the ALK, and the verification result is consistent with the 1.13 detection result. The detection device can successfully detect the gene fusion of the FFPE sample.
Industrial applicability
According to the invention, the device and the method for detecting the gene fusion of the FFPE sample are provided, which have the advantages of high detection speed, low resource requirement and high stability.
Claims (11)
1. An apparatus for detecting FFPE sample gene fusion, comprising the following modules:
the sequencing data acquisition module is used for acquiring the sequencing data of the FFPE sample;
a comparison module: the sequencing data acquisition module is connected with the sequencing data acquisition module and is used for comparing the acquired sequencing data with a reference sequence to acquire a comparison result, wherein the comparison result comprises soft shearing information and successful comparison information of the sequencing data;
And a re-comparison module: the sequencing fragment comparison module is connected with the comparison module and is used for comparing the sequencing fragment with the soft shearing information with the reference genome again to obtain a re-comparison result;
real integration breakpoint judgment module: the re-comparison module is connected with the sequencing fragment and is used for judging a real fusion breakpoint of the sequencing fragment; and
an output module: which is connected with the real fusion breakpoint judgment module and is used for outputting gene fusion detection results,
the real fusion breakpoint judgment module comprises the following sub-modules:
a filtering submodule: the re-comparison module is connected with the sequencing module and is used for filtering and removing sequencing fragments which are not successfully compared and sequencing fragments with low specific mass values;
a breakpoint information acquisition submodule: the sequencing sub-module is connected with the filtering sub-module and is used for searching sequencing fragments with the same fragment description information and acquiring breakpoint information;
fusing breakpoint screening submodules: the breakpoint information acquisition submodule is connected with the breakpoint information acquisition submodule and is used for screening fusion breakpoints in breakpoint information;
merging the break points and merging the sub-modules for the first time: the system is connected with the fusion breakpoint screening submodule and is used for merging the fusion breakpoints with the same breakpoint information into a real fusion breakpoint, and taking the number of the fusion breakpoints with the same breakpoint information as the support degree of the real fusion breakpoint, wherein the same breakpoint information refers to the condition that the chromosome number left _ chr of a breakpoint left sequence, the comparison position left _ pos of a first base at the breakpoint left side, the chromosome number right _ chr of the breakpoint right sequence and the comparison position right _ pos of the first base at the breakpoint right side are the same;
Merging the sub-modules again by fusing breakpoints: and the fusion breakpoint primary merging submodule is connected with the fusion breakpoint primary merging submodule and is used for merging the chromosome number left _ chr of the breakpoint left side sequence and the chromosome number right _ chr of the breakpoint right side sequence into a real fusion breakpoint, wherein the comparison position right _ pos of the first base at the right side of the breakpoint or the fusion breakpoint within a certain value of difference between the comparison positions left _ pos of the first base at the left side of the breakpoint.
2. The apparatus of claim 1, wherein the sequencing data is sequencing data obtained using a paired-end sequencing method.
3. The apparatus of claim 1, wherein the alignment module uses bwa software to find the corresponding position of the sequenced fragment in the reference sequence, obtain the soft-clip information and the successful alignment information of the sequenced fragment, and form a bam format file.
4. The apparatus of claim 1 or 3, wherein the realignment module comprises the following sub-modules:
length filter submodule: the sequencing fragment filtering module is connected with the comparison module and is used for filtering and removing sequencing fragments with the length smaller than a certain value in the sequencing fragments containing the soft shearing information;
a breakpoint judgment submodule: the length filtering submodule is connected with the length filtering submodule and is used for taking the joint of the part with the soft shearing information and the part with the normal comparison information in the sequencing fragment as a breakpoint according to the result data of the length filtering submodule;
Distinguishing sub-modules: the breakpoint judgment sub-module is connected with the soft cutting information part and the normal comparison information part, and is used for separating the soft cutting information part and the normal comparison information part at a breakpoint and respectively storing sequence information of the two parts into two files;
and then comparing the submodules: the distinguishing submodule is connected with the two files respectively storing the sequence information and the reference sequence and is used for comparing the two files respectively storing the sequence information with the reference sequence again to obtain a re-comparison result.
5. The apparatus of claim 4, wherein the re-alignment result comprises: description information qname, sequence information seq, comparison position POS and comparison quality value MAPQ of each sequencing fragment.
6. The apparatus of claim 4, wherein the re-alignment sub-module re-aligns the two fastq files using bwa software and forms a bam format file.
7. The apparatus of claim 4, wherein the length filtering submodule filters sequencing fragments with a length of less than 15-25 bp from the sequencing fragments containing soft-clip information.
8. The apparatus of claim 4, wherein the distinguishing sub-module saves the obtained sequence information to a fastq file.
9. The apparatus of claim 1, wherein the breakpoint information comprises:
left/right _ chr, chromosome number of the breakpoint left/right sequences;
left/right _ pos, alignment position of the first base left/right of the breakpoint;
left/right _ seq, sequence of base left/right of breakpoint;
sup, breakpoint support, number of sequencing fragments supporting the breakpoint.
10. The apparatus of claim 9, wherein the fusion breakpoint filter submodule comprises the following elements:
breakpoint quality filter element: the breakpoint A is used for filtering low-quality breakpoints, if the breakpoint A exists, the sup number of the breakpoint A is more than 5, the comparison quality values in the left _ seq and the right _ seq are both more than 30, and the mismatching rates are both less than 0.05, the breakpoint A is a fusion breakpoint; and
same breakpoint merge element: for merging the same break points, if there are break points a and B, left _ chr in a is equal to right _ chr in B, right _ chr in a is equal to left _ chr in B, left _ pos in a is equal to right _ pos in B, right _ pos in a is equal to left _ pos in B, then break points a and B are merged into a merged break point.
11. The apparatus of claim 9, wherein the merge break point reconsolidation submodule merges, according to the break point information, the merge break point a and the merge break point B into a real merge break point if there are a right _ pos at the merge break point a and a right _ pos at the merge break point B that are smaller than 5, and a left _ pos at the merge break point a and a left _ pos at the merge break point B that are smaller than 5.
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