CN106282163A - Construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection - Google Patents
Construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection Download PDFInfo
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Abstract
The invention discloses a kind of construction method based on Ion Torrent order-checking fusion of platforms gene test library and the method for fusion gene detection.Wherein, this construction method comprises the following steps: S1, sample DNA is carried out fragmentation, obtains DNA fragmentation；S2, carries out end reparation to DNA fragmentation and adds base A；S3, joint known to catenation sequence；S4, carries out PCR amplification, obtains sample nucleic acid library；S5, by based on target area capture platform for sample nucleic acid library probe hybrid capture fusion gene and the DNA fragmentation of chaperone；And S6, the product of S5 is introduced sequence measuring joints sequence by PCR, obtains fusion gene detection library.Application technical scheme, solves Ion Torrent order-checking platform in prior art and causes because RNA sample is defective to detect gene fusion and cannot detecting the unknown fused type problem merging breakpoint.
Description
Technical field
The present invention relates to biological technical field, in particular to one based on Ion Torrent order-checking fusion of platforms base
Construction method and the method for fusion gene detection because of detection library.
Background technology
Gene fusion belongs to the one of gene structure variation, refers to that the coding region of two or more gene joins end to end, in
Under same set of regulating and controlling sequence controls, the mosaic gene of composition.The expression product of fusion gene is fusion protein.
Along with constantly reducing and target area capture technique continuous of extensive parallel order-checking (secondary order-checking) cost
Maturation, gene test based on secondary sequencing technologies is more and more applied.Secondary order-checking compared with traditional detection method,
Having highly sensitive, accuracy is high, cheap, can detect the advantage of multiple target spot simultaneously.The order-checking of main flow is put down on the market
Platform mainly has two, is the order-checking of fluorescence reversibility cessation method and the Thermo Fisher company of Illumina company respectively
Semiconductor ion stream order-checking (Ion Torrent).
Target area capture utilizes sequence capturing technology the DNA of target area to be caught and is enriched with, in conjunction with high-flux sequence
Platform carries out the research strategy checked order.It is advantageous that compared with genome sequencing survey region is concentrated, and is substantially reduced order-checking
Expense, cycle and workload；It is applicable to high depth order-checking, finds low frequency sudden change.Capture mode mainly have two kinds, one be with
Thermo Fisher company is the amplicon capture technique based on multiplex PCR of representative, is set by the upstream and downstream in target area
Meter primer is by areas captured interested；Also having is exactly the solution hybridization capture technique with Agilent company as representative, this technology
By capturing for target area design cRNA or cDNA probe.The advantage of the method is made by probe and can capture
The flank of target area, such that it is able to the region that detection is merged with target area producer, therefore can detect at DNA level
Unknown gene fusion.
Ion AmpliseqTMIt is target area being captured based on multiplex PCR of Thermo Fisher company's exploitation
Technology.The amplicon that this technology uses in transcript profile aspect (rna level) in terms of detection fusion gene builds storehouse mode, and
And can only be according to the primer of specific position of fusion form design multiplex PCR.Therefore, for each sample, it is required for both carrying
Take DNA and extract again RNA.And clinical sample complexity is high, paraffin-embedded tissue (FFPE) sample fixed by particularly formalin,
Owing to the holding time is longer and preserving type is lack of standardization, cause the extraction comparison difficulty of nucleic acid.And RNA is easier to send out than DNA
Raw degraded, this has resulted in that many samples unsuccessfully cause owing to RNA extracts cannot the situation generation of detection fusion gene.
Summary of the invention
It is desirable to provide a kind of based on Ion Torrent order-checking fusion of platforms gene test library construction method and
The method of fusion gene detection, with solve in prior art Ion Torrent order-checking platform because of RNA sample defective causing cannot
Detect gene fusion and the unknown fused type problem merging breakpoint cannot be detected.
To achieve these goals, according to an aspect of the invention, it is provided one is flat based on Ion Torrent order-checking
The construction method in platform fusion gene detection library.This construction method comprises the following steps: S1, and sample DNA is carried out fragmentation,
To DNA fragmentation；S2, carries out end reparation to DNA fragmentation and 3 ' ends add base A；3 ' ends are connected and have the DNA sheet of A base by S3
Joint known to section end catenation sequence；S4, according to joint known to sequence, carries out PCR amplification, obtains sample nucleic acid library；
S5, by based on target area capture platform for sample nucleic acid library probe hybrid capture fusion gene and the DNA sheet of chaperone
Section, uses eluent to remove the nonspecific products introduced in acquisition procedure；And S6, the product of S5 is introduced Ion by PCR
Torrent Proton sequence measuring joints sequence, obtains based on Ion Torrent order-checking fusion of platforms gene test library.
Further, in S1, when sample DNA is less than 100ng, the vector rna of addition 500ng carries out auxiliary to be interrupted.
Further, S1 specifically includes: use ultrasound wave to interrupt instrument and sample DNA is broken into master tape at 150bp～300bp
DNA fragmentation.
Further, joint known to sequence is the sequence measuring joints of Illumina order-checking platform.
Further, in S6, the 5 ' ends of the PCR primer joint sequence containing Ion Torrent proton order-checking, 3 ' ends
With sequence known to joint have the overlapping sequence of 13bp.
Further, sample DNA fixes paraffin-embedded tissue from formalin.
According to another aspect of the present invention, it is provided that a kind of based on Ion Torrent order-checking fusion of platforms gene test
Method.The method comprises the following steps: use any of the above-described kind of construction method to build based on Ion Torrent order-checking fusion of platforms
Gene test library；And check order on the Proton of Ion Torrent checks order platform, analyze sequencing result and merged
The result of gene test.
Further, before checking order on the Proton of Ion Torrent checks order platform, Agilent is used
2100Bioanalyzer detects Insert Fragment size, and uses qPCR detection by quantitative library yield.
Further, analyze sequencing result to include: after filtering out the sequencing sequence containing " N " and low quality sequencing sequence, adopt
Carry out sequence alignment with BWA software and produce bam file, then use IGV software to carry out soft sequence of blocking to check.
Application technical scheme, Ion Torrent order-checking platform combines solution hybridization capture technique, based on target
The probe hybrid capture fusion gene of areas captured platform and the DNA fragmentation of chaperone are then flat with Ion Torrent order-checking
Platform checks order, and solves Ion Torrent order-checking platform in prior art and causes detecting gene fusion because RNA sample is defective
And the unknown fused type problem merging breakpoint cannot be detected.
Accompanying drawing explanation
The Figure of description of the part constituting the application is used for providing a further understanding of the present invention, and the present invention shows
Meaning property embodiment and explanation thereof are used for explaining the present invention, are not intended that inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows the structure side based on Ion Torrent order-checking fusion of platforms gene test library according to the present invention
The schematic flow sheet of method；
Fig. 2-1 and Fig. 2-2 shows that the fusion breakpoint IGV of the XHC sample CCDC6-RET of embodiment 1 checks that result is illustrated
Figure；
Fig. 3-1 and 3-2 shows that the IGV of the DYT sample TPM3-ROS1 fusion breakpoint of embodiment 1 checks result schematic diagram；
And
Fig. 4-1 and 4-2 shows that the IGV of the FSH sample EML4-ALK fusion breakpoint of embodiment 1 checks that result figure is illustrated
Figure.
Detailed description of the invention
It should be noted that in the case of not conflicting, the embodiment in the application and the feature in embodiment can phases
Combination mutually.Describe the present invention below with reference to the accompanying drawings and in conjunction with the embodiments in detail.
According to a kind of typical embodiment of the present invention, it is provided that a kind of based on Ion Torrent order-checking fusion of platforms gene
The construction method in detection library.As it is shown in figure 1, this construction method comprises the following steps: S1, sample DNA is carried out fragmentation,
To DNA fragmentation；S2, carries out end reparation to DNA fragmentation and 3 ' ends add base A；3 ' ends are connected and have the DNA sheet of A base by S3
Joint known to section end catenation sequence；S4, according to joint known to sequence, carries out PCR amplification, obtains sample nucleic acid library；
S5, by based on target area capture platform for sample nucleic acid library probe hybrid capture fusion gene and chaperone (partner
Gene) DNA fragmentation, uses eluent to remove the nonspecific products introduced in acquisition procedure；And S6, the product of S5 is led to
Cross PCR and introduce Ion Torrent Proton sequence measuring joints sequence, obtain based on Ion Torrent order-checking fusion of platforms gene inspection
Survey library.
Ion Torrent order-checking platform combines solution hybridization capture technique, and probe based on target area capture platform is miscellaneous
Hand over capture fusion gene and the DNA fragmentation of chaperone, then with Ion Torrent order-checking platform order-checking, solve existing skill
In art, Ion Torrent order-checking platform causes because RNA sample is defective to detect gene fusion and cannot detecting unknown fusion
The fused type problem of breakpoint.The capture library, target area using technical scheme to build can be at Ion
Check order on Torrent Proton sequenator, it is possible to stably output data, and positive sample can be merged from trace
In accurately detect target gene merge and provide correct fusion form.
Preferably, in S1, when sample DNA is less than 100ng, the vector rna (Carrier RNA) adding 500ng carries out auxiliary
Help and interrupt.If this is because sample DNA is less than 100ng, owing to DNA content is low, may be to sample when ultrasonic disruption
Do great damage, therefore add vector rna (Carrier RNA) and play the effect of protection sample DNA.
Preferably, S1 specifically includes: use ultrasound wave to interrupt instrument and sample DNA is broken into master tape 150bp's～300bp
DNA fragmentation.The DNA fragmentation of 150bp～300bp is applicable to the sequencing reading length of follow-up sequenator, the sequenator used in the present invention
For Ion Torrent proton, its sequencing reading length is single-ended 200bp, and therefore DNA is broken in this step 150bp-300bp length
It is the most suitable to spend.
According to a kind of typical embodiment of the present invention, joint known to sequence is that the order-checking of Illumina order-checking platform connects
Head.The purpose connecing known array joint is to introduce Ion Torrent according to known array design subsequent PCR primer
Proton sequence measuring joints.
The Y-shaped joint rather than the direct proton of interpolation that add Illumina before hybridization during library construction check order and connect
, there is following advantage in head: 1) avoids owing to proton sequence measuring joints is blunt end cloning, for trace sample, and can be due to
Joint efficiency deficiency causes the loss of sample DNA template, causes the loss of hereditary information；2) closed reagent that hybridization uses is avoided
The joint being required for proton redesigns, it is impossible to ensure sealing effect and capture rate；3) survey due to proton is avoided
In sequence joint, barcode sequence and Insert Fragment are apart from close, it is impossible to introduce barcode sequence by the means of PCR.
Preferably, in S6, PCR primer 5 ' end containing Ion Torrent proton order-checking joint sequence, 3 ' end with
Joint known to described sequence has the overlapping sequence of 13bp.It is to say, when designing primer, 5 ' ends of primer are containing Ion
The joint sequence of Torrent proton order-checking, 3 ' ends are containing the Iillumina sequence measuring joints complementary added with previous step
Sequence (13bp), PCR when, above-mentioned 13bp length can with Illumina sequence measuring joints sequence anneals renaturation, thus
Ion Torrent proton sequence measuring joints sequence is incorporated into the two ends of PCR primer.
In the building process in the front library of hybridization, with the addition of the sequence measuring joints of Illumina order-checking platform, so can cause
During PCR (introducing proton sequence measuring joints) after hybridization, the needs combined due to primer, need to retain a part
Illumina joint sequence, can cause if this partial sequence is the shortest PCR failure, oversize if data can be caused waste, process
Grope to determine afterwards and use the overlap sequence of 13bp to assist the introducing carrying out proton joint.
According to a kind of typical embodiment of the present invention, sample DNA fixes paraffin-embedded tissue from formalin.
According to a kind of typical embodiment of the present invention, it is provided that a kind of based on Ion Torrent order-checking fusion of platforms gene
The method of detection.The method comprises the following steps: uses the construction method of any of the above-described kind to build and checks order based on Ion Torrent
Fusion of platforms gene test library；And check order on the Proton of Ion Torrent checks order platform, analyze sequencing result
Obtain the result of fusion gene detection.
Application technical scheme, Ion Torrent order-checking platform combines solution hybridization capture technique, based on target
The probe hybrid capture fusion gene of areas captured platform and the DNA fragmentation of chaperone are then flat with Ion Torrent order-checking
Platform checks order, and solves Ion Torrent order-checking platform in prior art and causes detecting gene fusion because RNA sample is defective
And the unknown fused type problem merging breakpoint cannot be detected.
Preferably, before checking order on the Proton of Ion Torrent checks order platform, Agilent is used
2100Bioanalyzer detects Insert Fragment size, and uses qPCR detection by quantitative library yield.
According to a kind of typical embodiment of the present invention, analyze sequencing result and include: filter out the sequencing sequence containing " N "
(reads) with low quality reads after, use BWA software to carry out sequence alignment and produce bam file, then it is soft to use IGV software to carry out
Block reads to check.
Owing to Ion Torrent order-checking platform order-checking principle is limited, the order-checking error rate for tandem sequence repeats base is higher,
After order-checking completes, instrument automatic fitration can fall the sequencing sequence (reads) that sequencing quality is poor, and gene fusion is often sent out
Raw in tandem sequence repeats base, so can cause losing some key reads in initial data.For this problem,
The present invention uses the lookup the most original signal of telecommunication of order-checking to give the accuracy that the reads supporting to merge sudden change ensures to detect for change.
According to a kind of typical embodiment of the present invention, the method comprises the following steps:
The fragmentation of sample DNA the most to be detected
Use ultrasound wave to interrupt instrument (Covaris LE220R) according to table 1, sample is interrupted parameter to be broken into master tape and exist
The DNA fragmentation of 150bp～300bp；Especially, if sample DNA enters less than 100ng, the Carrier RNA that need to add 500ng
Row auxiliary interrupts.
Table 1
2.DNA fragment ends is repaired and 3 ' ends add A base；
3. the connection of joint
End connects joint known to the DNA random fragment end catenation sequence of A base, and (sequence is it is known that and rear
During continuous hybrid capture, there is the closed reagent for joint sequence can use).
4.PCR expands
According to known joint sequence primer, carry out PCR amplification, obtain sample nucleic acid library；
5. probe hybrid capture based on Agilent SureSelect target area capture platform, uses eluent to remove and catches
The nonspecific products introduced during obtaining；
6. introduce Ion Torrent Proton sequence measuring joints sequence by PCR
7. library detection
Use Agilent 2100Bioanalyzer detection Insert Fragment size；
Use qPCR detection by quantitative library yield.
Beneficial effects of the present invention is further illustrated, the technology being not described in the present invention below in conjunction with embodiment
Means can use ordinary skill in the art means to realize.
Embodiment 1
Patients with Non-small-cell Lung FFPE merges positive sample 3 example, sample ID, fusion gene and type, extracted amount and
Usage amount is shown in Table 2.
Table 2
The fragmentation of 1.DNA
Sonicator (Covaris LE220R) is used to be pressed by the Carrier RNA (500ng) of target dna and incorporation
According to specifically interrupting parameter and carry out the fragmentation of DNA, the parameter that interrupts of Covaris LE220R is arranged such as table 1.
DNA after interrupting carries out next step operation after the AMPure XP magnetic beads for purifying of 1.8X.
2. end reparation and 3 ' ends add A base
Reaction mixture is prepared, with rifle gently pressure-vaccum mixing up and down with reference to table 3 proportioning.
Table 3
Put into PCR instrument, program is set by table 4 and carries out reacting that (Gai Wen is set to 70 DEG C, and front 30min does not cover heat lid, and temperature arrives
65 DEG C, cover heat lid immediately).
Table 4
3. joint connects
Reaction mixture is prepared, with rifle gently pressure-vaccum mixing up and down according to the proportioning of table 5.
Table 5
It is divided into 2 pipes, often pipe 55ul, is put in PCR instrument, 20 DEG C of reaction 15min, close heat lid.After reaction terminates, use
The AMPure XP magnetic beads for purifying DNA sample of 0.8X.
4.PCR expands
Reaction mixture is prepared, with rifle gently pressure-vaccum mixing up and down according to the proportioning of table 6.
Table 6
Put into PCR instrument, program reaction be set by table 7..
Table 7
With the AMPure XP magnetic bead of 1.8X, amplified production is purified, finally library is dissolved in 30ul NF-water
In.Purified product is carried out Qubit quantitative
5. Library hybridization
5.1 by library according to the concentration value of previous step, take 600-700ng and be placed in concentrating instrument and be evaporated to 10ul, temperature sets
It is set to 45 DEG C, it is generally required to about 15-20min.
5.2 connect to the P5/P7 concentrating the QXT quick closure mixed liquor and each 1ul that add 5ul in complete sample
Head closed reagent (150uM), with of short duration centrifugal after pipettor piping and druming mixing, carries out next step.
5.3 follow-up hybridization and catching method are with reference to Agilent company SureSeclectQXTTest kit hybrid capture method.Press
Response procedures according to table 8 hybridizes 2h, suspends when three steps, in order to add probe.Use DynabeadsMyone
Streptavidin T1 (Streptavidin T1) magnetic bead captures, and uses eluent 1 (SureSelect Wash1) eluting one
Time, eluent 2 (SureSelect Wash2) eluting three times.
Table 8
6. after capture, library pcr amplification primer enters Ion Torrent Proton sequence measuring joints sequence
Reactant liquor is configured, with rifle gently pressure-vaccum mixing up and down according to table 9.
Table 9
Note: the * in the primer * that A end contains barcode is representative difference barcode sequence, Universal Primer P
It is shown in Table 3. with the sequence information of Barcode Primer A*
Put into PCR instrument, program is set by table 10 and reacts.
Table 10
With the AMPure XP magnetic bead of 1.8X, amplified production is purified, finally library is dissolved in 20ul NF-water
In.Purified product is carried out Qubit accurate quantification.
Primer specifying information in table 9 is shown in Table 10.
Table 10
Note: X is barcode sequence, and the sequence of corresponding barcode 1-12 is shown in Table 11.
Table 11
7. library detection
Purified product in step 6 is diluted to 2ng/ul, takes out 1ul and carry out the Agilent 2100Bioanalyzer (U.S.
Agilent company) detection, the library total length of structure is at about 300bp；Detect for qPCR, according to inspection it addition, further take out 1ul
Survey result and determine upper machine concentration.
According to the concentration of upper step gained, library is diluted to upper confidential ask after, check order flat at the Proton of Ion Torrent
Check order on platform, produce 400M data.
8. descend machine data results
Under data after machine, after filtering out the reads containing " N " and low quality reads (this area conventional parameter is arranged),
Use BWA software to carry out sequence alignment and produce bam file, then use IGV software to carry out the soft reads of blocking to check.Finally determine
Whether sample there occurs merges and merges the position occurred.Table 12 reflects the fusion detection knot of three samples in the present embodiment
Really, it is seen that this method can detect the fusion gene in positive sample and fused type accurately.Fig. 2-1 to 4-2 is each
The IGV of sample checks result.
Table 12
Fig. 2-1 to 4-2 is the graphical result using IGV software to detect above fusion gene, such as Fig. 2-1 and 2-2
Show is the testing result of XHC sample, and as can be seen from Fig., RET gene and two genes of CCDC6 have some sequences to have one
Part comparison is not gone up, and produces softclip sequence, and these softclip sequences can the reference sequences of the other side's gene in comparison.Institute
To be judged as the gene fusion (Fig. 2-1 to 4-2 for the comparison situation of display sequence, and the sequence of non-specific) of CCDC6-RET.
As can be seen from the above description, the above embodiments of the present invention achieve following technique effect:
Using the library constructing method that the present invention provides, can detect gene fusion at DNA level accurately, this is with existing
Ion Torrent banking process compare, have the advantage that
1) application technical scheme, Ion Torrent order-checking platform combines solution hybridization capture technique, based on mesh
The probe hybrid capture fusion gene of mark areas captured platform and the DNA fragmentation of chaperone, then check order with Ion Torrent
Platform checks order, and solves in prior art Ion Torrent order-checking platform and causes detecting gene because RNA sample is defective and melt
Close and cannot detect the unknown fused type problem merging breakpoint.And can be by single nucleotide mutation (SNV), insertion and deletion
(indel) and copy number variation (CNV) Joint Designing enter hybrid capture panel in, therefore can be at the bar not extracting RNA
Detect four kinds of common variation types under part simultaneously.And the Ion Ampliseq of routineTMTechnology must pass through two library (DNA
Library and RNA library) above four kinds of variation types could be detected completely, and gene cannot be detected when RNA extracts unsuccessful
Merge；
2) UNKNOWN TYPE can be detected merge.This method is melted due to the method that have employed liquid phase probe hybrid capture
Close the detection of gene, owing to probe captures, can be with the unknown fused type of detection fusion gene.And the Ion of routine
AmpliseqTMTechnology can only design amplimer according to known fused type, it is impossible to detection the unknown is merged the gene of breakpoint and melted
Close；
3) solution hybridization capture technique is applied at Ion Torrent order-checking platform by this method, combines Ion Torrent
The feature (can complete order-checking for about 3 hours) checking order fast of order-checking platform, substantially reduces the time of detection.
Present invention is directed at the library constructing method of Ion Torrent order-checking platform, can be at genome aspect (DNA water
Flat) the upper detection position that drives the fusion of gene and fusion gene to occur and form, thus instruct targeting medication to treat.Detection
Fusion gene include but not limited to the genes such as ALK, RET, ROS1.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for the skill of this area
For art personnel, the present invention can have various modifications and variations.All within the spirit and principles in the present invention, that is made any repaiies
Change, equivalent, improvement etc., should be included within the scope of the present invention.
Claims (9)
1. one kind based on Ion Torrent order-checking fusion of platforms gene test library construction method, it is characterised in that include with
Lower step:
S1, carries out fragmentation to sample DNA, obtains DNA fragmentation；
S2, carries out end reparation to described DNA fragmentation and 3 ' ends add base A；
3 ' ends are connected joint known to the DNA fragmentation end catenation sequence having A base by S3；
S4, according to joint known to described sequence, carries out PCR amplification, obtains sample nucleic acid library；
S5, by based on target area capture platform for described sample nucleic acid library probe hybrid capture fusion gene and chaperone
DNA fragmentation, use eluent to remove the nonspecific products introduced in acquisition procedure；And
S6, introduces Ion Torrent Proton sequence measuring joints sequence by the product of described S5 by PCR, obtain described based on
Ion Torrent checks order fusion of platforms gene test library.
Construction method the most according to claim 1, it is characterised in that in described S1, when described sample DNA is less than 100ng,
The vector rna of addition 500ng carries out auxiliary to be interrupted.
Construction method the most according to claim 1, it is characterised in that described S1 specifically includes: use ultrasound wave to interrupt instrument
Described sample DNA is broken into the master tape DNA fragmentation at 150bp～300bp.
Construction method the most according to claim 1, it is characterised in that joint known to described sequence is Illumina order-checking
The sequence measuring joints of platform.
Construction method the most according to claim 1, it is characterised in that in described S6,5 ' ends of PCR primer are containing Ion
The joint sequence of Torrentproton order-checking, joint known to 3 ' ends and described sequence has the overlapping sequence of 13bp.
Construction method the most according to claim 1, it is characterised in that described sample DNA fixes paraffin from formalin
The tissue of embedding.
7. a method based on Ion Torrent order-checking fusion of platforms gene test, it is characterised in that comprise the following steps:
The construction method as according to any one of claim 1 to 6 is used to build based on Ion Torrent order-checking fusion of platforms base
Because of detection library；And
Check order on the Proton of Ion Torrent checks order platform, analyze sequencing result and obtain the knot of fusion gene detection
Really.
Method the most according to claim 7, it is characterised in that carry out on the Proton of Ion Torrent checks order platform
Before order-checking, use Agilent 2100Bioanalyzer detection Insert Fragment size, and use qPCR detection by quantitative library to produce
Amount.
Method the most according to claim 7, it is characterised in that analyze described sequencing result and include: filter out the survey containing " N "
After sequence sequence and low quality sequencing sequence, use BWA software to carry out sequence alignment and produce bam file, then use IGV software to carry out
Soft sequence of blocking is checked.
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| CN108070910A (en) * | 2017-12-11 | 2018-05-25 | 上海赛安生物医药科技股份有限公司 | CfDNA captures banking process |
| CN108949905A (en) * | 2017-05-23 | 2018-12-07 | 深圳华大基因股份有限公司 | Compare library and its construction method |
| CN109161587A (en) * | 2018-09-26 | 2019-01-08 | 上海交通大学医学院附属上海儿童医学中心 | A method of detection chromosome repeated fragment broken site and location information |
| CN109576800A (en) * | 2018-12-07 | 2019-04-05 | 北京安智因生物技术有限公司 | A kind of construction method and its kit in the genetic test library of heredity dilated cardiomyopathy |
| CN110157785A (en) * | 2018-02-13 | 2019-08-23 | 浙江大学 | A kind of unicellular RNA sequencing library construction method |
| CN112342627A (en) * | 2019-08-09 | 2021-02-09 | 深圳市真迈生物科技有限公司 | Preparation method and sequencing method of nucleic acid library |
| CN112626206A (en) * | 2019-09-24 | 2021-04-09 | 深圳华大智造科技有限公司 | RNA fusion gene detection method and kit |
| CN113106149A (en) * | 2021-04-26 | 2021-07-13 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
| CN114015757A (en) * | 2021-08-31 | 2022-02-08 | 廖端芳 | Method for detecting fusion gene |
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| CN108949905A (en) * | 2017-05-23 | 2018-12-07 | 深圳华大基因股份有限公司 | Compare library and its construction method |
| CN108070910A (en) * | 2017-12-11 | 2018-05-25 | 上海赛安生物医药科技股份有限公司 | CfDNA captures banking process |
| CN107904666A (en) * | 2017-12-15 | 2018-04-13 | 中源协和基因科技有限公司 | A kind of library construction and quantitative approach applied to tumour driving genetic test |
| CN110157785A (en) * | 2018-02-13 | 2019-08-23 | 浙江大学 | A kind of unicellular RNA sequencing library construction method |
| CN109161587A (en) * | 2018-09-26 | 2019-01-08 | 上海交通大学医学院附属上海儿童医学中心 | A method of detection chromosome repeated fragment broken site and location information |
| CN109576800A (en) * | 2018-12-07 | 2019-04-05 | 北京安智因生物技术有限公司 | A kind of construction method and its kit in the genetic test library of heredity dilated cardiomyopathy |
| CN112342627A (en) * | 2019-08-09 | 2021-02-09 | 深圳市真迈生物科技有限公司 | Preparation method and sequencing method of nucleic acid library |
| CN112626206A (en) * | 2019-09-24 | 2021-04-09 | 深圳华大智造科技有限公司 | RNA fusion gene detection method and kit |
| WO2022067936A1 (en) * | 2020-09-30 | 2022-04-07 | 厦门飞朔生物技术有限公司 | Method for constructing library for detecting fgfr1/2/3 fusion gene of cholangiocarcinoma on basis of high-throughput sequencing |
| CN113106149A (en) * | 2021-04-26 | 2021-07-13 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
| CN113106149B (en) * | 2021-04-26 | 2023-01-17 | 福建和瑞基因科技有限公司 | Preparation method of fusion gene detection reference substance and application of fusion gene detection reference substance |
| CN114015757A (en) * | 2021-08-31 | 2022-02-08 | 廖端芳 | Method for detecting fusion gene |
| CN114015757B (en) * | 2021-08-31 | 2024-02-02 | 江门市灿明生物科技有限公司 | Method for detecting fusion gene |
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