CN107699957A - Fusion based on DNA, which is quantitatively sequenced, builds storehouse, detection method and its application - Google Patents
Fusion based on DNA, which is quantitatively sequenced, builds storehouse, detection method and its application Download PDFInfo
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Abstract
The invention discloses a kind of fusion based on DNA to be quantitatively sequenced banking process, and step includes：1) genomic DNA fragment；2) DNA fragmentation sorts；3) unidirectional specific PCR amplification, obtains the amplified production with position of fusion；4) single-stranded universal libraries joint sequence is connected at 3 ' ends of amplified production sequence；5) universal PC R is expanded, and is enriched with fusion region segments, purified, quantitative, obtains sequencing library.The invention also discloses method, the purposes of this method that fusion sequencing detection is carried out by above method constructed dna library, and the unidirectional specific primer group of fusion for above-mentioned banking process.The present invention passes through unidirectional specific amplification DNA fragmentation, fusion sequence is captured, then NGS sequencing DNA libraries are obtained by being enriched with, not only increases the ratio of the valid data of capture, and the speed for building storehouse and detection is accelerated, it is suitable for FFPE samples or liquid biopsy.
Description
Technical field
The present invention relates to field of gene detection, the sequencing more particularly to fusion detects, and is to be related to more specifically
The preparation in the DNA sequencing library of fusion, and detected using the high-flux sequence of DNA sequencing library progress fusion
Method.
Background technology
Being detected from first fusion (BCR/ABL1) of early stage the 1980s confirms, fusion has turned into more
The important symbol of individual cancer kind, such as：BCR/ABL1 is used to detect chronic myelogenous leukemia；EML4/ALK is used to detect malign lung
Gland cancer；TMPRSS2/ERG is used to detect prostate cancer, etc..
21 century, with the rise of targeted drug, cancer treatment drugs, especially non-small cell lung cancer (NSCLC)
Medicine, also begin to use the metabolic pathway where fusion.Permit to be used for for example, 2011 years grams of azoles obtain FDA for Buddhist nun
The treatment of ALK and ROS1 Gene Fusions；Ai Le in 2015 permits the treatment for ALK gene fusion for Buddhist nun's acquisition FDA；Suo Lafei
Buddhist nun and Sutent obtain the treatment license of RET Gene Fusions.
In today that malignant tumour is precisely detected and personalized treatment concept is suggested, in order to avoid medicine contraindication and tolerance
Cause futile treatment, delay the treatment time of preciousness, turn into clinician's administration for effective specific detection of fusion
Important evidence.The fusion detection of early stage is on the basis of tradition organizes biopsy, using FISH, chromosome bar
With analysis and RT-PCR analytical technologies, to confirm the tumour cell of patient, whether there occurs the Gene Fusion of specific gene.But
Following defect be present in detection of the cytology and Protocols in Molecular Biology of these early stages for fusion：1) detection time is long；
2) false positive rate is higher；3) detection technique requires high, it is necessary to which skilled experienced testing staff, is unfavorable for standardizing；4) melting
In the case of conjunction gene type cell is poor in cancerous tissue, can not effectively it detect.With NGS (high-flux sequence) technology
It is increasingly mature, and enter clinical gene detection field comprehensively, above mentioned problem has obtained effective solution.NGS detection techniques have
Sensitivity, specificity are high, and detection sample DNA requirement is few, and technology maturation, the series of advantages such as quick easy to operate,
Under its development that technology application cost reduces and flux constantly increases, NGS technologies turn into the Mainstream Platform of fusion detection.
For being to extract the RNA of tissue samples with sequencing or PCR method detection fusion gene, mode the most direct, lead to
Reverse transcription technology synthesis cDNA is crossed, the extron merged is captured and is completed to analyze.Such mode is based in genome
DNA levels, the intron sequences region between the exon sequence that the merging points of two genes all merges, and this region can
Energy>1kb, even reading longer generation sequencing technologies, also can not effectively cover that (reading of NGS sequencing technologies, which is grown, typically to exist
100-400bp), can not Direct Analysis fusion include subregion；The position of the merging point of some Gene Fusions only exists
Have in COSMIC databases report just more than 20, can not confirm merge position position sample, it is also not possible to by special
PCR is used to be sequenced to be enriched with the sequence of fusion position, and the both sides sequence of the ripe fusion RNA after shearing is then
Know, the sequence that enrichment length is less than 200bp can be expanded by PCR, for NGS high-flux sequences.Based on above reason, mesh
The fusion sequencing detection technique of preceding main flow is based on RNA.But introducing and its advantage with liquid biopsy concept
Property, the fusion detection method based on RNA has highlighted its limitation.
Liquid Biopsy, refer to only extract patient blood plasma or urine in dissociate Tumour DNA (ctDNA,
Circulating tumor DNA), by gene trapping come quick detection tumor cell gene type.In tumour cell
In life cycle, it may occur that cell death (many reasons of generation, here is omitted), so as to which internal nucleic acid substances be discharged
Out, and enter the circulatory system or excretory system, there also is the nucleic acid of healthy cell certainly, every milliliter of blood under regular situation
The yield of dissociative DNA is 5-20ng in slurry, and in so a small amount of dissociative DNA, ctDNA content may only have 0.1-5%, and just
Because the application of highly sensitive NGS technologies, make it possible detection ctDNA.It can be seen that liquid Biopsy compares traditional group
Knitting biopsy has a variety of advantages：1) patient is encroached on small；2) can multiple sampling monitoring；3) without carrying out pathology differentiation to sampling.
Therefore, the detection technique of various tumour cells is all improved one after another, with suitable for liquid biopsy.
However, compared to ctDNA, ctRNA degree of fragmentation is even more serious, typically in 10-25bp (ctDNA fragments
Change degree is typically in 150-200bp), therefore, ctRNA is not appropriate for the detection for fusion under the prior art, and this makes
Fusion detection that must be based on RNA is difficult to be applied in liquid biopsy.
In addition, the fusion detection based on RNA also faces, most of patient is beyond affordability repeatedly to puncture sampling, can only
The problem of obtaining pathological examination remaining FFPE (formalin fixation FFPE) sample.FFPE samples are fixed due to formaldehyde
Reason, RNA therein have occurred that serious degraded, it is difficult to provide the RNA fragments for the sufficient length that detection needs.
At present, the fusion sequence measurement based on DNA is by capturing fusion with probe in genomic library
Region identified, is purpose is strong the advantages of this method, can be saved data volume and cost；Shortcoming is easily de-
Target, when dissociative DNA is detected especially in liquid biopsy, valid data ratio is low, it is necessary to improve starting amount of DNA and sequencing data
Amount, so as to cause cost high, operating time length, operation difficulty is high, or even influences detection.
The content of the invention
One of the technical problem to be solved in the present invention, which is to provide a kind of fusion based on DNA and is quantitatively sequenced, builds storehouse side
Method, it builds, and the storehouse time is short, and cost is low, can be applied to liquid biopsy.
In order to solve the above technical problems, banking process is quantitatively sequenced in the fusion based on DNA of the present invention, including it is following
Step：
1) sample genomic dna fragmentation, and fragmentation products are purified；
2) DNA fragmentation after purification is sorted, the DNA fragmentation of length needed for acquisition；
3) unidirectional specific PCR amplification is carried out to sorting gained DNA fragmentation, obtains the amplified production with position of fusion,
The amplified production is purified；
4) single-stranded universal libraries joint sequence is connected on 3 ' ends of the sequence of amplified production obtained by step 3), and to even
Thing of practicing midwifery is purified；
5) universal PC R amplifications are carried out, are enriched with fusion region segments, and amplified production is purified, be quantitative, are obtained
DNA library is sequenced in fusion.
The step 2) can sort the DNA fragmentations of 350bp after purification with 2% agarose gel electrophoresis.
The step 3), pcr amplification reaction system include：μ l, the Taq polymerizations of 2 × Phusion HF PCR mixed solutions 25
Enzyme 1 μ l, DMSO 1 μ l, the μ l of fusion specific primer mixture 2, DNA fragmentation sorting the μ l of product 15, the μ l of nuclease-free water 6,
The μ l of cumulative volume 50；Pcr amplification reaction condition is：99 DEG C of holding 2min；99 DEG C of 15s, 60 DEG C of 2min, totally 30 circulations；10 DEG C of guarantors
Hold.It is SEQ ID NO to include sequence in the fusion specific primer mixture:1~20 primer.
The step 4), universal libraries joint sequence such as SEQ ID NO:Shown in 21.
The step 5), pcr amplification reaction system include：The μ of 2 × Phusion HF PCR mixed solutions, 25 μ l, FB primer 1
The μ l of l, P1B amp 1, connect the μ l of purified product 23, the μ l of cumulative volume 50；Pcr amplification reaction condition is：94 DEG C of holding 2min；94℃
30s, 50 DEG C of 30s, 68 DEG C of 30s, totally 2 circulations；98 DEG C of 30s, 62 DEG C of 30s, 68 DEG C of 30s, totally 14 circulations；68 DEG C of holdings
5min.Wherein, FB primers are selected from SEQ ID NO:Sequence shown in 22~30；P1B amp sequence such as SEQ ID NO:31 institutes
Show.
The second technical problem to be solved by the present invention is to provide one group of specific amplification for being used for above-mentioned banking process and merged
The primer of gene generation area, the primer sets include SEQ ID NO:Sequence shown in 1~20.
The third technical problem to be solved by the present invention, which is to provide a kind of fusion based on DNA and is quantitatively sequenced, builds storehouse reagent
Box, the kit include the DNA library prepared with above-mentioned banking process.
The four of the technical problem to be solved in the present invention are to provide a kind of detection method of the fusion based on DNA, the party
The high-flux sequence that the DNA library that method is prepared using above-mentioned banking process carries out fusion detects.
The five of the technical problem to be solved in the present invention are to provide the detection method of the above-mentioned fusion based on DNA in liquid
Application in biopsy.
The fusion sequencing library construction method based on DNA of the present invention, after by sample genomic dna fragmentation,
By unidirectional specific amplification, fusion sequence is obtained, universal linker sequence is reconnected, passes through the universal linker sequence and specificity
Universal sequence on unidirectional amplimer, purpose region fragment is enriched with, amplification obtains NGS sequencing DNA libraries, compared to existing
DNA banking process, there is advantages below and beneficial effect：
1. using single primer extend capture purpose region, upstream is opened without random primer or universal linker sequence in capture
Beginning can avoids background influence caused by universal primer, disclosure satisfy that the requirement more than 50% valid data substantially, highest can
Up to the 70% of reading, solve the problems, such as that the existing fusion detection based on RNA is difficult to the RNA for obtaining appropriate quality, can
Applied to FFPE samples or liquid biopsy.
2. detection is quick, cost is low, and two-step pcr is obtained with sequencing library, it is not necessary to numerous and diverse probe acquisition procedure,
Whole Library development flow can foreshorten to 6-8 hours from 3 days, and cost reduces by 50%, is adapted to the needs of clinical quick detection.
3. applied widely, all ALK pattern of fusion genes are suitably adapted for, can detect Co-factor propagation type or known pattern of fusion
Location breakpoint.
4. needed to use due to the other types abrupt climatic change of tumour cell and DNA, therefore without extracting RNA respectively
And DNA, so as to save time and cost.
Brief description of the drawings
Fig. 1 is the Gene Fusion reads distribution maps of the embodiment of the present invention.
Embodiment
More specifically understand to have to technology contents, feature and effect of the present invention, in conjunction with specific embodiment, to this hair
Bright technical scheme is further described in detail.
Instrument equipment, experiment material and reagent are as follows in embodiment：
First, instrument
AB ProFlex PCR instruments, IKA MS3Digital vortex oscillators, the fluophotometers of IVGN Qubit 3.0,
IKA Mini G palm centrifuges；
2nd, material
Rainin pipettors (μ l of specification 10,20,200,1000), AXYGEN common filters joint (Universal Fit
Filter Tips, μ l of specification 10,20,200,1000), DYNAL DynaMagTM-2magnet magnetic frames, Ambion
The low absorption PCR light-wall pipes of the transparent flat cover of Magnetic Stand-96, AXYGEN 0.2ml, the colourless low absorption centrifuge tubes of AXYGEN
(1.5ml、2.0ml)；
3rd, reagent
Analyze pure absolute ethyl alcohol, IVGNDsDNA HS kits, NEB T4DNA ligases, NEB Phusion surpass
Fidelity PCR reactant mixtures, BECKMANXP、NEBDsDNA fragmentation reagents.
The experimental method of unreceipted actual conditions in embodiment, generally according to normal condition, such as Sambrook et al., divide
Son clone：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) described in
Condition, or according to the condition proposed by manufacturer.
The construction method of ALK (anaplastic lymphoma kinase) fusion sequencing library of the present embodiment, idiographic flow are：
1. genomic DNA fragment
1) following component is mixed in the centrifuge tube on ice：(DNA extracts from patient FFPE, blood plasma or living tissue to DNA, uses
Measure as 1-16 μ l, total amount 200-500ng), the μ l of 10X fragmentations reaction buffer 2, add water to supply 18 μ l.
2) be vortexed concussion 3 seconds, of short duration centrifugation, is placed on ice.
3) NEB of whirlpool concussion bottleDsDNA fragmentation reagents 3 seconds, of short duration centrifugation, 2 μ l are taken to be added to above-mentioned
In the centrifuge tube of step 1), whirlpool shakes 3 seconds and of short duration centrifugation.
4) fragmentation reaction is carried out, response procedures are：37 DEG C 20 points (FFPE samples are 15 minutes), 4 DEG C of holdings.
5) of short duration centrifugation, place on ice.
2.DNA fragmentation products purify
1) 80 μ l nuclease-free waters are added in DNA fragmentation product, are then transferred in the low adsorption tubes of 1.5ml, add 120 μ l
(1.2 times of volumes)Mixing 5 times is played in AMPure XP magnetic beads, suction, and room temperature is placed 5 minutes.
2) reaction tube is placed in 5 minutes or until solution clarification, supernatant discarding on magnetic frame.
3) 70% ethanol of 500 μ l Fresh is added, reaction tube is gently moved up and down close to magnetic frame, to clean magnetic
Pearl, then suck supernatant.This step is repeated once, to ensure that ethanol is fully sucked.
4) reaction tube is maintained on magnetic frame, drying at room temperature 5 minutes, removed from magnetic frame, add 20 μ l free nucleic acids
Enzyme water, vortex vibration 3s.
5) reaction tube is placed on magnetic frame, stands 2min, supernatant is drawn to new reaction tube, for follow-up DNA pieces
Section sorting.
3.DNA fragments sort
With the DNA fragmentations of 2% agarose gel electrophoresis sorting 350bp after purification.
4.PCR is expanded
Pcr amplification reaction system includes：The μ l of 2 × Phusion HF PCR mixed solutions 25, Taq polymerase 1 μ l, DMSO
(dimethyl sulfoxide (DMSO)) 1 μ l, the μ l of fusion specific primer mixture 2, DNA fragmentation sorting product 15 μ l, the μ of nuclease-free water 6
L, the μ l of cumulative volume 50.
Include following primer in the fusion specific primer mixture：
(1)GGCCAAGGCGGGCTCTGTGCACTCACCAATCATGA(SEQ ID NO:1)
(2)GGCCAAGGCGGGAGCTTCCGTTTTGGCTTGGCCTG(SEQ ID NO:2)
(3)GGCCAAGGCGCACCATCGTGATGGACACTGAAGGA(SEQ ID NO:3)
(4)GGCCAAGGCGCTATCTCTCTGCCTGGAGGGTGGTG(SEQ ID NO:4)
(5)GGCCAAGGCGGCAGTAAGAGCTGGTTGGGACCACA(SEQ ID NO:5)
(6)GGCCAAGGCGCTACCGGCAGATCCCTTTGCCTGCA(SEQ ID NO:6)
(7)GGCCAAGGCGGCCGAGCACGTAGTAACCATGCAAC(SEQ ID NO:7)
(8)GGCCAAGGCGCGCTATGTGCTCAGTTCCCTCCTCT(SEQ ID NO:8)
(9)GGCCAAGGCGGCAAACTCCATGGAAGCCAGAACAA(SEQ ID NO:9)
(10)GGCCAAGGCGCCCACCCTCTAGGGTTGTCAATGAA(SEQ ID NO:10)
(11)GGCCAAGGCGACAGGACCTCTTTGGACTGCAGTTT(SEQ ID NO:11)
(12)GGCCAAGGCGCCAAGCCACAGAGTTGGAGAAGAGC(SEQ ID NO:12)
(13)GGCCAAGGCGCTGTGCTCAGCCATTGGGTAGGGCA(SEQ ID NO:13)
(14)GGCCAAGGCGGTGCCATGGAGCCTAAGGAAGTTTC(SEQ ID NO:14)
(15)GGCCAAGGCGCAGGGAAGGCTGGGTGAACCAGCAG(SEQ ID NO:15)
(16)GGCCAAGGCGGGCAGAGTCATGTTAGTCTGGTTCC(SEQ ID NO:16)
(17)GGCCAAGGCGTCCTCCCCTGAGCTCTGAACCTTTC(SEQ ID NO:17)
(18)GGCCAAGGCGAGCTCCATCTGCATGGCTTGCAGCT(SEQ ID NO:18)
(19)GGCCAAGGCGAGGGCGCTCACCGAATGAGGGTGAT(SEQ ID NO:19)
(20)GGCCAAGGCGTTTGTGGCTAGAGGAGTCTGCGGTG(SEQ ID NO:20)
Pcr amplification reaction condition is：99 DEG C of holding 2min；99 DEG C of 15s, 60 DEG C of 2min, totally 30 circulations；10 DEG C of holdings.
5.PCR amplified productions purify
1) 60 μ l (1.2 times of volumes) are added in pcr amplification productAMPure XP magnetic beads, vortex vibration
3s, room temperature are placed 5 minutes.
2) reaction tube is placed in 5 minutes or until solution clarification, supernatant discarding on magnetic frame.
3) freshly prepared 70% ethanol of 500 μ l is added, reaction tube is gently moved up and down close to magnetic frame, to clean magnetic
Pearl, then suck supernatant.This step is repeated once, to ensure that ethanol is fully sucked.
4) reaction tube is maintained on magnetic frame, drying at room temperature 5 minutes, removed from magnetic frame, add 23 μ l free nucleic acids
Enzyme water, vortex vibration 3s.
5) reaction tube is placed on magnetic frame, stands 2min, drawn 22 μ l supernatants to new reaction tube, connected for joint
Reaction.
6. joint connects
μ l of T4 ligase buffer solutions 5, P1B- are added in the reaction tube of the above-mentioned pcr amplification product containing 22 μ l after purification
ADP2 μ l, μ l of T4 ligases 1, the μ l of nuclease-free water 20, amount to 50 μ l.Inhaled with pipettor and play mixing 5 times, be incubated at room temperature 30min.
Product can be in -20 DEG C of preservations.
P1B-ADP sequences are：phos-ATCACCGACTGCCCATAGAGAGGCTGAGAC-NH2C6(SEQ ID NO:21)
7. joint connection product purifies
1) 60 μ l (1.2 times of volumes) are added in joint connection productAMPure XP magnetic beads, vortex vibration
3s, room temperature are placed 5 minutes.
2) reaction tube is placed in 5 minutes or until solution clarification, supernatant discarding on magnetic frame.
3) freshly prepared 70% ethanol of 500 μ l is added, reaction tube is gently moved up and down close to magnetic frame, to clean magnetic
Pearl, then suck supernatant.This step is repeated once, to ensure that ethanol is fully sucked.
4) reaction tube is maintained on magnetic frame, drying at room temperature 5 minutes, removed from magnetic frame, add 24 μ l free nucleic acids
Enzyme water, vortex vibration 3s.
5) reaction tube is placed on magnetic frame, stands 2min, drawn 23 μ l supernatants into new 0.2ml PCR pipes, use
In following amplification reaction.
8.PCR is expanded
Pcr amplification reaction system includes：(X is what is selected to 2 × Phusion HF PCR mixed solutions 25 μ l, FB X
Barcode code 1~8,13) the μ l of 1 μ l, P1B amp 1, connect the μ l of purified product 23, the μ l of cumulative volume 50.
FB X sequences are following (underline for barcode sequences)：
FB1:CCATCTCATCCCTGCGTGTCTCCGACTCAGCTAAGGTAACGGCCAAGGCG(SEQ ID NO:22)
FB2:CCATCTCATCCCTGCGTGTCTCCGACTCAGTAAGGAGAACGGCCAAGGCG(SEQ ID NO:23)
FB3:CCATCTCATCCCTGCGTGTCTCCGACTCAGAAGAGGATTCGGCCAAGGCG(SEQ ID NO:24)
FB4:CCATCTCATCCCTGCGTGTCTCCGACTCAGTACCAAGATCGGCCAAGGCG(SEQ ID NO:25)
FB5:CCATCTCATCCCTGCGTGTCTCCGACTCAGCAGAAGGAACGGCCAAGGCG(SEQ ID NO:26)
FB6:CCATCTCATCCCTGCGTGTCTCCGACTCAGCTGCAAGTTCGGCCAAGGCG(SEQ ID NO:27)
FB7:CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCGTGATTCGGCCAAGGCG(SEQ ID NO:28)
FB8:CCATCTCATCCCTGCGTGTCTCCGACTCAGTTCCGATAACGGCCAAGGCG(SEQ ID NO:29)
FB13:CCATCTCATCCCTGCGTGTCTCCGACTCAGTCTAACGGACGGCCAAGGCG(SEQ ID NO:30)
P1B amp sequences are:GTCTCAGCCTCTCTATGGGCAGT(SEQ ID NO:31)
Pcr amplification reaction condition is：94 DEG C of holding 2min；94 DEG C of 30s, 50 DEG C of 30s, 68 DEG C of 30s, totally 2 circulations；98℃
30s, 62 DEG C of 30s, 68 DEG C of 30s, totally 14 circulations；68 DEG C of holding 5min.
9.PCR amplified productions purify
1) 25 μ l are added in the system after pcr amplification reaction terminatesAMPure XP magnetic beads, vortex vibration
3s, room temperature place 5min.
2) reaction tube is placed on magnetic frame 5 minutes or until solution is clarified, by supernatant, (supernatant is the library piece that needs
Section) suck in new reaction tube.
3) 25 μ l are added in the supernatant of absorptionAMPure XP magnetic beads, vortex vibration 3s, room temperature are placed
5min。
4) reaction tube is placed in 5 minutes or until solution clarification, supernatant discarding on magnetic frame.
5) 70% ethanol of 500 μ l Fresh is added, reaction tube is gently moved up and down close to magnetic frame to clean magnetic bead,
Then supernatant is sucked.This step is repeated once, to ensure that ethanol is fully sucked.
6) reaction tube is maintained on magnetic frame, drying at room temperature 5 minutes, removed from magnetic frame, add 50 μ l free nucleic acids
Enzyme water, vortex vibration 3s, centrifuges 2s.
5) reaction tube is placed on magnetic frame, stands 2min, drawn supernatant to new reaction tube, quantified for QUBIT.
The quantitative libraries of 10.QUBIT
1) useDsDNA HS kits quantitative sample DNA on Qubit fluophotometers.
2) in a 1.5ml centrifuge tube, 597 μ l Qubit dsDNA HS buffer solutions and 3 μ l Qubit dsDNA are merged
HS reagents, vortex oscillation mix, and are prepared into Qubit working solutions.
3) 3 Qubit test tubes of mark respectively：#1 standards QC, #2 standards QC, #3 sample cells；In #1 standards QC and #
In 2 standard QCs, 190 μ l Qubit working solutions are added, then respectively by Qubit standard HS#1 and #2 standard items 10
μ l are added in corresponding #1 standards QC and #2 standard QCs, and vortex oscillation mixes；199 μ l Qubit are added in #3 sample cells
Working solution and 1 μ l are through isolating and purifying the sample DNA being recovered to (PCR after being previously used for quantitative purified of QUBIT
Amplified production), vortex oscillation mixes.
4) after standing 2 minutes, Qubit is opened, DNA measure is selected, re-calibrates in #1 standards QC and #2 standard QCs
Standard items.
5) #3 sample cells are put into reading in Qubit to quantify, and calculate the quantitative values using 1 μ l samples.
6) by product dilution to 22ng/ml (100pM), the DNA available for Ion Torrent platform high-flux sequences is obtained
Library.
Above-mentioned DNA library feeding Ion Torrent high-flux sequences instrument is subjected to sequencing detection.
The principle of Gene Fusion detection method is：(each read comes from one to the unimolecule reads being sequenced using two generations
Single-molecule DNA sequence), if same reads (comprising both-end and single-ended) two parts compare the difference of genome respectively
Position, and be not repeat (repetition) region, then this read may be the potential reads merged.Last root
According to cutoff>100, filter out the fusion and its pairing gene of the positive.Comprise the following steps that：
1) quality control is carried out to the reads of all primitive sequencers using fastqc, then removes joint sequence.
2) up-to-standard sequence is taken, software is compared with BWA, is compared with hg19 genome chromosomes.
3) reads of the improper comparison on target gene (ALK) is filtered out.
4) by the reads of improper comparison, fasta forms are changed into, is compared onto genome, found out using blat softwares
All possible matched position, maping is with the position of gene in hg19, and calculates number.
5) according to reads number>100, and remove the repetition section that several genomes often occur, check base with IGV
Because of fusion reads distributions, filter out the fusion of the positive and its match gene.
Testing result shows that EML4 is merged (shown in Figure 1) with ALK gene.
Sequence table
<110>Lead star biotechnology（Shanghai）Co., Ltd
<120>Fusion based on DNA, which is quantitatively sequenced, builds storehouse, detection method and its application
<130> CPC-NP-17-100462
<160> 31
<170> PatentIn version 3.3
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ggccaaggcg ggctctgtgc actcaccaat catga 35
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ggccaaggcg ggagcttccg ttttggcttg gcctg 35
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<221> misc_feature
<223>Primer
<400> 3
ggccaaggcg caccatcgtg atggacactg aagga 35
<210> 4
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 4
ggccaaggcg ctatctctct gcctggaggg tggtg 35
<210> 5
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 5
ggccaaggcg gcagtaagag ctggttggga ccaca 35
<210> 6
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 6
ggccaaggcg ctaccggcag atccctttgc ctgca 35
<210> 7
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 7
ggccaaggcg gccgagcacg tagtaaccat gcaac 35
<210> 8
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 8
ggccaaggcg cgctatgtgc tcagttccct cctct 35
<210> 9
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 9
ggccaaggcg gcaaactcca tggaagccag aacaa 35
<210> 10
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 10
ggccaaggcg cccaccctct agggttgtca atgaa 35
<210> 11
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 11
ggccaaggcg acaggacctc tttggactgc agttt 35
<210> 12
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 12
ggccaaggcg ccaagccaca gagttggaga agagc 35
<210> 13
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 13
ggccaaggcg ctgtgctcag ccattgggta gggca 35
<210> 14
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 14
ggccaaggcg gtgccatgga gcctaaggaa gtttc 35
<210> 15
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 15
ggccaaggcg cagggaaggc tgggtgaacc agcag 35
<210> 16
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 16
ggccaaggcg ggcagagtca tgttagtctg gttcc 35
<210> 17
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 17
ggccaaggcg tcctcccctg agctctgaac ctttc 35
<210> 18
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 18
ggccaaggcg agctccatct gcatggcttg cagct 35
<210> 19
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 19
ggccaaggcg agggcgctca ccgaatgagg gtgat 35
<210> 20
<211> 35
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 20
ggccaaggcg tttgtggcta gaggagtctg cggtg 35
<210> 21
<211> 30
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Joint
<400> 21
atcaccgact gcccatagag aggctgagac 30
<210> 22
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 22
ccatctcatc cctgcgtgtc tccgactcag ctaaggtaac ggccaaggcg 50
<210> 23
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 23
ccatctcatc cctgcgtgtc tccgactcag taaggagaac ggccaaggcg 50
<210> 24
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 24
ccatctcatc cctgcgtgtc tccgactcag aagaggattc ggccaaggcg 50
<210> 25
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 25
ccatctcatc cctgcgtgtc tccgactcag taccaagatc ggccaaggcg 50
<210> 26
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 26
ccatctcatc cctgcgtgtc tccgactcag cagaaggaac ggccaaggcg 50
<210> 27
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 27
ccatctcatc cctgcgtgtc tccgactcag ctgcaagttc ggccaaggcg 50
<210> 28
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 28
ccatctcatc cctgcgtgtc tccgactcag ttcgtgattc ggccaaggcg 50
<210> 29
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 29
ccatctcatc cctgcgtgtc tccgactcag ttccgataac ggccaaggcg 50
<210> 30
<211> 50
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 30
ccatctcatc cctgcgtgtc tccgactcag tctaacggac ggccaaggcg 50
<210> 31
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<221> misc_feature
<223>Primer
<400> 31
gtctcagcct ctctatgggc agt 23
Claims (10)
1. banking process is quantitatively sequenced in the fusion based on DNA, it is characterised in that comprises the following steps：
1) sample genomic dna fragmentation, and fragmentation products are purified；
2) DNA fragmentation after purification is sorted, the DNA fragmentation of length needed for acquisition；
3) unidirectional specific PCR amplification is carried out to sorting gained DNA fragmentation, the amplified production with position of fusion is obtained, to this
Amplified production is purified；
4) single-stranded universal libraries joint sequence is connected on 3 ' ends of the sequence of amplified production obtained by step 3), and connection is produced
Thing is purified；
5) universal PC R amplifications are carried out, are enriched with fusion region segments, and amplified production is purified, be quantitative, are merged
Gene sequencing DNA library.
2. according to the method for claim 1, it is characterised in that step 2), sub-elected with 2% agarose gel electrophoresis
The DNA fragmentations of 350bp after purification.
3. according to the method for claim 1, it is characterised in that step 3), pcr amplification reaction system include：2×
The μ l of Phusion HF PCR mixed solutions 25, Taq polymerase 1 μ l, DMSO 1 μ l, the μ l of fusion specific primer mixture 2,
DNA fragmentation sorts the μ l of product 15, the μ l of nuclease-free water 6, the μ l of cumulative volume 50；Pcr amplification reaction condition is：99 DEG C of holding 2min；
99 DEG C of 15s, 60 DEG C of 2min, totally 30 circulations；10 DEG C of holdings.
4. the method according to claim 1 or 3, it is characterised in that step 3), the sequence of the primer sets of pcr amplification reaction
Such as SEQ ID NO:Shown in 1~20.
5. according to the method for claim 1, it is characterised in that step 4), the universal libraries joint sequence such as SEQ ID
NO:Shown in 21.
6. according to the method for claim 1, it is characterised in that step 5), pcr amplification reaction system include：2×
The μ l of 25 μ l, FB primer of Phusion HF PCR mixed solutions, 1 μ l, P1B amp 1, connect the μ l of purified product 23, the μ l of cumulative volume 50；
The FB primers are selected from SEQ ID NO:Sequence shown in 22~30；The sequence of the P1B amp such as SEQ ID NO:Shown in 31；
Pcr amplification reaction condition is：94 DEG C of holding 2min；94 DEG C of 30s, 50 DEG C of 30s, 68 DEG C of 30s, totally 2 circulations；98 DEG C of 30s, 62 DEG C
30s, 68 DEG C of 30s, totally 14 circulations；68 DEG C of holding 5min.
7. the primer sets of specific amplification fusion generation area, it is characterised in that the sequence of the primer sets such as SEQ ID
NO:Shown in 1~20.
8. the fusion based on DNA, which is quantitatively sequenced, builds storehouse kit, it is characterised in that the kit includes claim 1-
DNA library prepared by any one of 6 methods describeds.
9. detection method is quantitatively sequenced in the fusion based on DNA, it is characterised in that usage right is required described in any one of 1-6
DNA library prepared by method carries out the high-flux sequence detection of fusion.
10. application of the detection method in liquid biopsy is quantitatively sequenced in fusion described in claim 9.
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| CN112912514A (en) * | 2018-10-17 | 2021-06-04 | 珀金埃尔默保健科学（澳大利亚）私人有限公司 | Barcoding of nucleic acids |
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| CN110564745B (en) * | 2019-08-27 | 2021-07-20 | 杭州市第一人民医院 | Lung cancer rare ALK fusion mutant gene and detection primer, kit and detection method thereof |
| CN110484655A (en) * | 2019-08-30 | 2019-11-22 | 中国医学科学院病原生物学研究所 | The detection method of two generation of parainfluenza virus full-length genome sequencing |
| CN110484655B (en) * | 2019-08-30 | 2021-03-30 | 中国医学科学院病原生物学研究所 | Detection method for parainfluenza virus whole genome second-generation sequencing |
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| CN112852918A (en) * | 2021-01-20 | 2021-05-28 | 深圳百人科技有限公司 | Two-step PCR technique |
| CN114015757A (en) * | 2021-08-31 | 2022-02-08 | 廖端芳 | Method for detecting fusion gene |
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