CN112795654A - Method and kit for organism fusion gene detection and fusion abundance quantification - Google Patents
Method and kit for organism fusion gene detection and fusion abundance quantification Download PDFInfo
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Abstract
The invention discloses a method and a kit for detecting fusion genes of organisms and quantifying fusion abundance, belonging to the technical field of molecular biology.
Description
Technical Field
The invention relates to a method and a kit for detecting fusion genes of organisms and quantifying fusion abundance in the technical field of molecular biology.
Background
Gene Fusion (Gene Fusion) refers to the process of connecting the coding regions of two or more genes end to end, placing them under the control of the same set of regulatory sequences (including promoter, enhancer and terminator) to form a chimeric Gene, usually breaking itself and then joining with another Gene, leading the 3 'end Gene containing the coding region of kinase domain to fuse with the 5' end of various heterologous upstream partner genes, recombining into a new Gene (Fusion Gene). Gene fusions are typically due to chromosomal rearrangements. Aberrant gene fusion events are associated with the development of tumors, and thus alterations in these genes are important diagnostic, prognostic, and predictive biomarkers.
For fusion assays, the current gold standard for clinical gene fusion assays is Fluorescence In Situ Hybridization (FISH). Although FISH sensitivity is high, there are: only one of the known fusions can be detected, and the disadvantages of large sample usage and poor specificity are overcome. RNA nucleic acid directly reflects the splicing condition of exons of a fusion gene and a fusion partner gene, is the first choice material for clinical test of the fusion gene at present, is spliced by exons based on RNA level in companies at present, detects fusion by using an RT-PCR method, commonly uses ARMS-PCR or digital PCR, and has the advantages of high sensitivity and good specificity, but has small flux, and only can detect a plurality of known fusions at one time. If it is desired to cover several types of fusion that are common in tumors, a large amount of RNA nucleic acid is required, which is contrary to the amount of microRNA that FNA and the like puncture tissues. With the rapid development of high throughput sequencing technology, the means for detecting fusion genes based on NGS technology is also gradually applied and accepted. At present, a detection kit based on a second-generation sequencing platform on the market is commonly established by a probe capture method based on DNA level, but the cost is high and the operation flow is complicated; the RNA level reverse transcription and multiple PCR technology combines the sensitivity and specificity of qPCR and the intuition of the NGS technical result, has simple and convenient operation and short time consumption, can detect all fusion types by only one sample, and has outstanding methodological advantages.
However, most of the existing technical solutions are to reverse RNA into cDNA and then construct a library with cDNA (patent numbers: CN111088365A, CN110241215A, and CN109371139A), which not only increases the experimental operation process and increases the risk of pollution, but also has relatively less information for detecting fusion genes and limited clinical use. The fusion positive threshold of the prior art (patent No. CN 104894271A) is defined according to the reading value, and the method is extremely unstable and is easily interfered by various factors such as the quantity of sequencing data, methodology and the like.
The molecular tag is a molecular marker for counting which is obtained by connecting each original nucleic acid fragment in a sample or connecting each original nucleic acid fragment to a nucleic acid fragment by methods such as anchoring extension, template displacement extension, RACE and the like, so that each original nucleic acid fragment in the sample has a unique tag. The fusion ratio in the original RNA can be accurately quantified by subsequently distinguishing different original molecules through respective labels of the wild type and the fusion type. The method can stably and accurately trace back to the original fusion positive state, and can also carry out absolute quantification on the abundance of the fusion transcript through counting of the molecular tag, thereby solving the existing fusion detection problem.
Disclosure of Invention
The invention relates to a method for detecting organism fusion genes and quantifying fusion abundance after template amplification by using a template-dependent primer extension strategy, and a kit for detecting by using the method. By the technical means, the detection and the auxiliary diagnosis of clinical tumor lesions are realized.
The invention is realized by the following technical scheme:
a method for detecting the fusion gene of living body and quantitatively determining the fusion abundance includes such steps as reverse transcription and amplification of the nucleic acid of living body, using the reverse transcription primer extension strategy, reverse transcription and amplification of template, detecting the fusion gene of living body, and quantitatively determining the fusion abundance. The method is characterized in that: the organism fusion gene detection comprises the steps that at least one reverse transcription primer carries out reverse transcription reaction under the action of reverse transcriptase; amplifying by using 5 ' end specific forward primers of various heterologous upstream partner genes, and any two primers of a3 ' end specific gene reverse primer containing a kinase domain coding region, a sequencing joint in a Tn5 transposition complex, a forward primer at one end of a sequencing platform specificity or a 5 ' end of a reverse primer with the other end, wherein the amplification is single primer extension, isothermal amplification or PCR amplification; introducing a specific sequence at the 5' end of the template-dependent forward primer or the template-dependent reverse primer; introducing a specific sequence into Tn5 transposition complex; or a specific sequence is introduced into the 5' end of the forward primer at one specific end of the sequencing platform or the reverse primer with the other end; the specific sequence is a molecular marker, and the molecular marker comprises a molecular label; displaying, calculating and quantifying the abundance of the fusion transcript by using a molecular marker method; presenting the fusion result and quantifying the abundance of the fusion transcript by using a molecular biology research method of second-generation sequencing, third-generation sequencing, fluorescent quantitative PCR, digital PCR, a gene chip, mass spectrometry, capillary electrophoresis or Sanger sequencing; the technical scheme is further explained as follows: the reverse transcriptase has RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and terminal nucleotidyl transferase activity, the terminal transferase activity is only shown when the reverse transcriptase contacts the 5 'end of an RNA template, additional nucleotides are added to the end of cDNA 3', the length of the added nucleotide sequence is 1-3nt, the sequence is any one of Poly (A), Poly (G), Poly (C) and Poly (T), and the additional added nucleic acid is also called template-free dangling; the cDNA3 'end is added with additional nucleotides to complementarily match with complementary bases, such as a primer containing any one of Poly (A), Poly (G), Poly (C) and Poly (T) at its 3' end, and 1-3nt in length, which can initiate template displacement reaction, such primer containing PCR primer anchor site (i.e., template displacement primer) to perform PCR amplification in combination with reverse transcription primer. In addition, if a single terminal transferase is used after the reverse transcription is finished, more template-free overhangs with the length of 1-300nt can be added, and the template-free overhangs can also be complementarily paired with the template displacement primers so as to carry out PCR amplification by combining with the reverse transcription primers; the technical scheme is further explained as follows: the reverse transcription primer is oligo d (T), and a random primer or a single-ended template-dependent primer generates a single-stranded cDNA; the primer for template replacement is a primer containing a molecular label and common use of a sequencing platform, the 3' end is a base which is vertically complementary to the template-free overhang at the end of a strand, and the length of the primer is 1-50nt, wherein the base is at least one of Poly (A), Poly (G), Poly (C) and Poly (T); the above reverse transcription primers (or a combination thereof) may be combined with a template displacement primer to perform amplification; the technical scheme is further explained as follows: designing a template-dependent forward primer by the upstream chaperone gene 10-500nt away from the fusion breakpoint and 5'; the 3' end contains a gene design template-dependent reverse primer of a kinase domain coding region, and the length of the primer is 15-50 nt; the technical scheme is further explained as follows: the molecular marker is a sequence with specific length, the length of the molecular marker is 2-200nt, the sequence is a random base N (A/G/C/T) or a semi-random base M (A/C), V (A/C/G), R (A/G), H (A/C/T) and W (A/T)
D, (A/G/T), S, (C/G), B, (C/G/T), Y, (C/T) and K, (G/T), or the combination of a random base and a fixed base, or the combination of a semi-random base and a random base; the technical scheme is further explained as follows: the molecular marker is added to a sequencing joint in a template-dependent forward primer, a template-dependent reverse primer, a Tn5 transposition complex, a forward primer at one specific end of a sequencing platform or a reverse primer with the other end, or is added to any two amplification primers; the technical scheme is further explained as follows: the source of the organism is one or more than one of fresh tissue, fixed tissue, FFPE slices, paraffin rolls, paraffin sample blocks, blood, plasma, serum, saliva, body fluid, urine, milk, follicular fluid and semen, and the nucleic acid template is nucleic acid of the organism, including Total RNA, mRNA, miRNA, long-chain non-coding RNA or DNA; the technical scheme is further explained as follows: the template amplification is directly carried out by taking DNA as a substrate, or Total RNA as a substrate, or mRNA, miRNA and lncRNA as substrates, preferably Total RNA as a substrate, and can be single-stranded RNA, RNA/DNA hybrid strand or double-stranded cDNA; the technical scheme is further explained as follows: before amplification is carried out by taking Total RNA as a substrate or mRNA as a substrate, reverse transcription or in vitro transcription is required, preferably, reverse transcription is carried out; the technical scheme is further explained as follows: the cDNA generated by reverse transcription is single-stranded or double-stranded; the length of the cDNA is 20-100000 nt; the technical scheme is further explained as follows: the template amplification is one of single primer extension, isothermal amplification, multiple displacement amplification or PCR amplification; the technical scheme is further explained as follows: the product amplified by the reaction template can be used as fluorescent quantitative PCR or digital PCR; the technical scheme is further explained as follows: the product of the amplified reaction template is a second-generation sequencing library and a third-generation sequencing library; the technical scheme is further explained as follows: the product of the amplified reaction template is a pre-machine product of a gene chip, mass spectrometry, capillary electrophoresis or Sanger sequencing method; the technical scheme is further explained as follows: the organism is an organism with reference genome information, preferably a human or a mouse; the technical scheme is further explained as follows: the organism fusion gene detection is used for detecting tumor fusion genes and identifying the abundance of fusion transcripts; the tumors include solid tumors and hematological tumors; the technical scheme is further explained as follows: the tumor fusion gene detection is performed on human tumor fusion genes and comprises RNA extraction, reverse transcription, amplification primer amplification, purification and addition of an NGS library building joint; the technical scheme is further explained as follows: converting RNA into cDNA through reverse transcription in the fusion reverse transcription reaction, performing PCR amplification on a target sequence by using a specific primer with a molecular tag, performing two-round PCR enrichment amplification after an amplification product is purified and enriched, and finally purifying to obtain a sequencing library; the technical scheme is further explained as follows: the RNA reverse transcription and PCR amplification reaction is completed in a single tube; a method and a kit for detecting organism fusion genes and quantifying fusion abundance are characterized in that: the detection process comprises the following steps:
1) extraction of RNA: extracting RNA from a tissue or blood sample using an RNA extraction kit;
2) reverse transcription and amplification of specific fragments: reverse transcription and a first round of PCR (amplification of a specific target fragment) reaction are completed in a single tube, RNA is taken as a template, a fused 3' end gene reverse primer with a molecular tag is taken as a reverse transcription specific primer, and cDNA is obtained; amplifying the cDNA by using a specific multiplex PCR primer and an amplification reagent to obtain a specific target fragment;
3) and (3) purifying a product: adding quantitative magnetic beads into the PCR amplification product obtained in the last step for purification, and removing RNA and residual amplification primers;
4) connecting a sequencing joint: adding a specific label and a joint reagent in an NGS library establishing reagent into the purified product obtained in the previous step, and performing PCR amplification to obtain a library fragment;
5) library detection: quantifying the library prepared in the last step by using a Qubit, performing quality inspection by using an Agilent Bioanalyzer 2100 or an instrument with the same function, and detecting the concentration of the library and the fragment size of the library to ensure that the final library fragment is in the range of 220-250 bp;
6) and (3) machine sequencing: cyclizing the library qualified in the previous step, and performing high-throughput sequencing by using a sequencing platform;
1) analyzing the data result by bioinformatics analysis software;
the technical scheme is further explained as follows: the bioinformatics analysis software comprises a computer medium of data, comprising the following analysis steps, in particular:
1) according to Q30, the sequencing quality of the base is firstly evaluated, and the molecular tag sequences in the sequencing sequence are mapped and counted. Duplification determination based on the diversity of the molecular tag sequences (sequences with the same tag are considered to be derived from the same initial RNA template, which is amplified during PCR);
2) firstly, comparing the Reads with a reference genome or transcriptome, removing a repeated molecular tag, screening out the number of the Reads which accords with the corresponding Split Reads (the Reads containing a breakpoint of two gene fusions) and the number of wild-type Reads of the 3' end gene of the trans-intron region, and calculating: n (a)SplitReads)/[(N(Split Reads)+N(wild type Reads)]And further obtaining the fusion ratio of the RNA in the original state.
The invention has the beneficial effects that:
the invention adopts a multiple PCR capture technology, the method can stably and accurately trace back to the original fusion positive state, and can also absolutely quantify the abundance of the fusion transcript by counting the molecular tags, thereby solving the existing fusion detection problem; transcription and amplification reaction of a target region are completed in one tube, so that the detection accuracy is greatly improved, and the additional tube opening/pipetting risk is reduced.
Drawings
The accompanying drawings, which are a part of the specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 detailed library construction principles of the present application.
FIG. 2 the principle of construction of the CCDC6-RET fusion library in a preferred embodiment of the present application.
FIG. 3 TBP amplification efficiency curves in a preferred embodiment of the present application.
Figure 4 TBP dissolution profile in a preferred embodiment of the present application.
FIG. 5 HMBS amplification efficiency curves in a preferred embodiment of the present application.
Fig. 6 HMBS dissolution profile in a preferred embodiment of the present application.
FIG. 7 LMNA amplification efficiency curves in a preferred embodiment of the present application.
Figure 8 LMNA dissolution profile in a preferred embodiment of the present application.
FIG. 9 fragments of a library in a preferred embodiment of the present application.
Detailed Description
The technical means adopted by the present invention and the effects thereof are further explained below with reference to the drawings and the examples, and the technical solutions of the present invention are further explained by the specific embodiments, but the present invention is not limited within the scope of the examples.
A tumor gene fusion checkpoint as shown in table 1 below for fusion genes and fusion types:
TABLE 1 fusion genes and fusion types
Material
1. Tissue cell RNA extraction kit (centrifugation column), Rui JING organism, Cat RJ 001T-A;
2.2XKAPA 2G Fast Multiplex Mix, Kapa biosystems, cat # KK 5802;
HiScript II Reverse Transcriptase (Reverse Transcriptase), Vazyme, cat # R201-02;
agencourt AMPure XP (purified magnetic beads), Beckman, cat # A63881;
a Qsep-100 nucleic acid fragment analyzer;
6. huada sequencers MGISEQ-200 and MGISEQ-2000
The materials used in the following examples are not limited to those listed above, and other similar materials may be substituted, and those skilled in the art should understand that the materials and equipment used are conventional and the equipment is not specifically defined, or the equipment is recommended by the manufacturer.
Example 1 the primer sequences for amplifying the mutation sites of tumor-associated genes were synthesized by primer synthesis company, Shanghai.
The RNA primer design principle of the present invention is described in detail below by taking CCDC6-RET as an example. As shown in figure 1, the 5 'end CCDC6 gene exon 1 is fused with 3' RET gene exon 12 by using the position of the break point as a boundary, so that the upstream primer is designed on the CCDC6 gene exon 1, and the downstream primer is designed on the RET gene exon 12; the RET wild type upstream primer is designed on the No. 11 exon, and the downstream is consistent with the fusion gene downstream primer.
(1) RNA specific primers whose primer sequences are shown in Table 2:
TABLE 2 fusion genes and their primer sequences
Wild type gene and primer sequence thereof
Molecular tag with P representing phosphorylated NNNNNNNN representing 6 bases
The primer content in the reaction solution is as follows: mu.L of each 10. mu.mol/L primer was 0.4. mu.L.
(2) Primer of housekeeping gene
The method is a database building process through housekeeping gene quality control. Screening low-expression genes TBP and HMBS respectively in tumors by consulting documents, expressing genes LMNA, designing primers for each gene in adjacent exons spanning introns respectively, and requiring intron length to be more than 1000bp for preventing DNA pollution; designing multiple pairs of primers for each gene, verifying the amplification efficiency and the melting curve of each pair of primers by QPCR, and finally screening out the primers with the amplification efficiency (Eff%) ranging from 95% to 115% and only a single melting curve (FIGS. 2-6), wherein the primer lists are shown in Table 3:
TABLE 3 housekeeping genes and their primer sequences
Example 2: pretreatment of samples and nucleic acid extraction
The qualified professional samples with a needle, the sample comprising greater than 1mg of the punctured tissue sample. After completion of the biopsy, the tissue was completely infiltrated in RNA storage solution as soon as possible. Samples were stored at-20 ℃ prior to sample shipment.
RNA in the sample was extracted using a tissue cell RNA extraction kit (centrifugal column method) by referring to the kit instructions.
1. Mechanical tissue sample homogenization:
the sample is placed in a suitable glass tube or a centrifuge tube, 350. mu.L of Buffer RL and DTT mixed lysate (100:1) is added, a probe is inserted into the lysate, and the sample is homogenized intermittently at a high speed for 15-20 seconds each time until the sample is completely homogenized.
2. Extraction of nucleic acids after homogenization
About 350 μm L of the homogenized sample was centrifuged at 14000rpm for 3min and the supernatant was transferred to a centrifuge tube and kept ready for use.
2.1 Total RNA extraction
2.1.1 adding 70% ethanol with the same volume to the supernatant after centrifugation, and sucking and beating for 3-5 times by using a pipette gun.
2.1.2 transfer the mixture to Hipure RNA Mini Columns I, which are loaded into collection tubes, and centrifuge at 12000rpm for 1 min.
2.1.3 discard waste, put the column back into the collection tube, add 350. mu.L Buffer RW1 to the column, centrifuge at 12000Rpm for 1 min.
2.1.4 discard the waste liquid and put the column back into the collection tube. 50U DNase I enzyme was added to the center of the membrane of the column. Standing at room temperature for 15 min.
2.1.5 Add 500. mu.L Buffer RW1 to the column, let stand for 3min, centrifuge at 12000rpm for 1 min.
2.1.6 discard the waste and put the column back into the collection tube. Add 500. mu.L Buffer RW2 to the column and centrifuge at 12000rpm for 1 min.
2.1.7 discard the waste and put the column back into the collection tube. Add 500. mu.L of Buffer RW2 to the column. Centrifuge at 12000rpm for 1 min.
2.1.8 discard the waste liquid and return the column to the collection tube. The column was emptied at 14000rpm for 5 min.
2.1.9 the RNA columns were packed in 1.5ml centrifuge tubes. Add 15-30. mu.L RNase Free Water to the center of the membrane of the column. Standing at room temperature for 1 min. Centrifuge at 12000rpm for 1 min. RNA samples were stored at-80 ℃.
Example 3: construction of the library
The library establishing process of the kit is as follows:
(1) RNA reverse transcription and first round PCR amplification (amplification of a target region) take RNA as a template and a fused 3' end gene reverse primer with 6 random bases as a reverse transcription specific primer to obtain cDNA; amplifying cDNA with specific multiplex PCR primer and amplification reagent to obtain specific target segment, and reaction in a single tube. The following systems were prepared using reverse transcriptase from Vazyme and its KAPA multi-pool Mix:
the PCR instrument is set with the following conditions for reaction:
(2) first round PCR product purification
1) After the programmed reaction is finished, centrifuging the sample for a short time, adding non-nucleic acid water to the sample to make up for 20 mu L, transferring the sample to XP magnetic beads containing 14 mu L (0.7X), mixing uniformly by vortex, and incubating for 2min at room temperature;
2) placing the sample on a magnetic frame, after the solution is clarified, transferring the supernatant into XP magnetic beads containing 10 mu L (0.5 x), uniformly mixing by vortex, and incubating for 5min at room temperature;
3) placing the sample on a magnetic frame, and removing the supernatant after the solution is clarified;
4) adding 200 mu L of freshly prepared 80% ethanol into the sample, rotating the sample on a magnetic frame for one circle, and after 30S, removing the ethanol; repeating the step 1 time and 2 times in total;
5) centrifuging the sample, discarding residual liquid on a magnetic frame, uncovering and drying the magnetic beads for 1-2min until the magnetic beads do not reflect light;
6) adding 9 mu L of nucleic-Free Water into the magnetic beads, uniformly mixing by vortex, and centrifuging for a short time;
7) transferring the sample with the magnetic beads into a new PCR tube, and directly carrying out second round amplification without removing the magnetic beads;
(3) second round of amplification
The purified product was subjected to a second round of amplification using sequencing adapter primers and Index primers, and the reactions were formulated on a clean bench according to the following system:
different sample libraries used different Index numbered primers
The PCR instrument is set with the following conditions for reaction:
(4) second round PCR product purification
1) Transferring 20 μ L of the sample with beads to 20 μ L (1X) XP beads, vortexing, and incubating at room temperature for 5 min;
2) placing the sample on a magnetic frame, and removing the supernatant after the solution is clarified;
3) adding 200 mu L of freshly prepared 80% ethanol into a sample, rotating the sample on a magnetic frame for one circle, and after 30S, removing the ethanol; repeating the step 1 time and 2 times in total;
4) centrifuging the sample tube, discarding residual liquid on a magnetic frame, uncovering and drying the magnetic beads for 1-3min until the surfaces of the magnetic beads are not reflected;
5) adding 15 mu L of nucleic-Free Water into the magnetic beads, uniformly mixing by vortex, and incubating for 5min at room temperature;
6) placing the sample tube on a magnetic frame, transferring the supernatant to a new clearly-marked 1.5mL EP tube after clarification, wherein the purified product is a constructed library;
(5) detection of the library: the constructed library is accurately quantified by using the Qubit, quality inspection is carried out by using Qsep-100, and the fragment size of the library is detected to ensure that the library fragment is in the range of 220-250bp (figure 8).
(6) And (3) machine sequencing: performing high-throughput sequencing on the library qualified in the step (5) by using a Huada sequencer after cyclization by using a Huada cyclization kit;
(7) and (4) performing data result analysis by using bioinformatics analysis software. According to Q30, firstly, the sequencing quality of the base is evaluated, and simultaneously, the random base sequence at the front end of the sequencing sequence is cut and combined into the sequence ID; duplification determination was performed based on the random tag sequence added to the ID position, and sequences aligned to the same position and having compatible tags were considered to be derived from the same starting RNA template. Firstly, comparing the Reads with a reference genome, screening Split Reads (Reads containing two gene fusion breakpoints) meeting corresponding parameters and 3' end gene-spanning wild type Reads, and calculating the ratio of the Split Reads to the sum of the Split Reads and the wild type Reads so as to obtain the fusion proportion of RNA in the original state.
Example 4 Performance verification
1. And (3) sensitivity analysis: the fusion ratio of 1% detectable in RNA samples was determined by using a standard of 5% positive cells, and performing gradient dilution of 2%, 1%, and 0.5%.
2. And (3) repeatability experiment: different initial amounts of RNA were added for each reaction, and high-throughput sequencing was repeated 10 times, with consistent results for 10 tests, and a compliance rate of 100%.
3. Clinical sample validation
30 clinical thyroid gland puncture tissue samples of Shanghai Ruiki hospital are collected, the kit is compared with a digital PCR detection result, the consistency of the detection results of the kit and the digital PCR detection result is 100%, and specific results are shown in Table 4. But the kit can simultaneously and rapidly detect the fusion of multiple genes, saves the detection cost and improves the detection efficiency, thereby having good clinical application value
TABLE 430 comparison of different methodologies for tissue sample puncture
The embodiments show that the kit can be used for simultaneously detecting multiple genes and multiple sites related to thyroid cancer, has the characteristics of high sensitivity and good specificity, and can meet the existing clinical requirements.
The kit adopts a multiple PCR capture technology and an NGS sequencing technology, is used for qualitatively detecting various variations of CCC6-RET, NCOA4-RET, PAX8/PPARG and ETV6-NTRK3 fusion genes in a tumor tissue sample, and provides help for the auxiliary diagnosis and treatment of tumors by detecting fusion sites and combining clinical pathological analysis results.
The kit is a one-round PCR amplification by designing a specific primer to carry out reverse transcription and amplification on a target sequence, carrying out two-round PCR enrichment amplification on an amplification product after purification and enrichment of magnetic beads, and finally obtaining a sequencing library through purification of the magnetic beads.
The kit can more accurately and stably detect RNA analysis fusion by designing a molecular label and screening out a specific housekeeping gene.
In addition to the high-throughput multiplex PCR capture technology of the present invention, the fusion detection can be performed by digital PCR or ARMS-PCR, but the number of loci detected by the two is small. Therefore, this method is currently most suitable when multiple gene multiple sites are required for simultaneous detection.
The kit adopts a multiple PCR capture technology, respectively designs primers aiming at CCC6-RET, NCOA4-RET, PAX8-PPARG and ETV6-NTRK3, specifically screens low-expression housekeeping genes TBP and HMBS in tumors, and expresses housekeeping genes LMNA.
The kit RNA is built by using gene specific primers with molecular labels, reverse transcription and amplification reaction of a target region are completed in one tube, so that the detection accuracy is greatly improved, and the extra tube opening/pipetting risk is reduced.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit it. The basic principles and the main features of the invention have been described above with specific embodiments, on the basis of which some modifications or alterations can be made without departing from the essence of the corresponding technical solution.
Sequence listing
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gaacgacatg gctacgatcc gacttcaggg cagctatgcc tcct 44
<210> 7
<211> 42
<212> DNA
<213> Unknown (Unknown)
<400> 7
gaacgacatg gctacgatcc gacttgcctt tccccatgct gc 42
<210> 8
<211> 46
<212> DNA
<213> Unknown (Unknown)
<400> 8
gaacgacatg gctacgatcc gacttgcact acccagaggc ctatgc 46
<210> 9
<211> 47
<212> DNA
<213> Unknown (Unknown)
<400> 9
gaacgacatg gctacgatcc gacttgcaac ctctcgactc accagac 47
<210> 10
<211> 51
<212> DNA
<213> Unknown (Unknown)
<400> 10
tcctaagacc gcttggcctc cgacttnnnn nnccaaagtt ggtgggccag a 51
<210> 11
<211> 44
<212> DNA
<213> Unknown (Unknown)
<400> 11
gaacgacatg gctacgatcc gacttatggt ctctgtctcc ccgc 44
<210> 12
<211> 50
<212> DNA
<213> Unknown (Unknown)
<400> 12
gaacgacatg gctacgatcc gacttctgga aactctatac acacacagcc 50
<210> 13
<211> 58
<212> DNA
<213> Unknown (Unknown)
<400> 13
tcctaagacc gcttggcctc cgacttnnnn nngtgagttg atgggactag atgatctc 58
<210> 14
<211> 51
<212> DNA
<213> Unknown (Unknown)
<400> 14
tcctaagacc gcttggcctc cgacttnnnn nngtgggctg gctgagtcct c 51
<210> 15
<211> 46
<212> DNA
<213> Unknown (Unknown)
<400> 15
gaacgacatg gctacgatcc gactttcagc tactcctctt ccggtg 46
<210> 16
<211> 54
<212> DNA
<213> Unknown (Unknown)
<400> 16
tcctaagacc gcttggcctc cgacttnnnn nncaagaacc aagttcttcc gagg 54
<210> 17
<211> 49
<212> DNA
<213> Unknown (Unknown)
<400> 17
gaacgacatg gctacgatcc gacttggaga ttctcctatt gacccagaa 49
<210> 18
<211> 51
<212> DNA
<213> Unknown (Unknown)
<400> 18
tcctaagacc gcttggcctc cgacttnnnn nnccaaagtt ggtgggccag a 51
<210> 19
<211> 46
<212> DNA
<213> Unknown (Unknown)
<400> 19
gaacgacatg gctacgatcc gacttactcg catccctgtc attgag 46
<210> 20
<211> 58
<212> DNA
<213> Unknown (Unknown)
<400> 20
tcctaagacc gcttggcctc cgacttnnnn nngtgagttg atgggactag atgatctc 58
<210> 21
<211> 49
<212> DNA
<213> Unknown (Unknown)
<400> 21
gaacgacatg gctacgatcc gacttggttc tcttcgtcat gatcaacaa 49
<210> 22
<211> 51
<212> DNA
<213> Unknown (Unknown)
<400> 22
tcctaagacc gcttggcctc cgacttnnnn nngtgggctg gctgagtcct c 51
<210> 23
<211> 25
<212> DNA
<213> Unknown (Unknown)
<400> 23
gaacgacatg gctacgatcc gactt 25
<210> 24
<211> 43
<212> DNA
<213> Unknown (Unknown)
<400> 24
tgtgagccaa ggagttgttg tcttcctaag accgcttggc ctc 43
<210> 25
<211> 48
<212> DNA
<213> Unknown (Unknown)
<400> 25
gaacgacatg gctacgatcc gacttgggag ctgtgatgtg aagtttcc 48
<210> 26
<211> 57
<212> DNA
<213> Unknown (Unknown)
<400> 26
tcctaagacc gcttggcctc cgacttnnnn nnggagaaca attctgggtt tgatcat 57
<210> 27
<211> 45
<212> DNA
<213> Unknown (Unknown)
<400> 27
gaacgacatg gctacgatcc gacttgcaga gaaagttccc gcatc 45
<210> 28
<211> 52
<212> DNA
<213> Unknown (Unknown)
<400> 28
tcctaagacc gcttggcctc cgacttnnnn nncactgaac tcctgctgct cg 52
<210> 29
<211> 45
<212> DNA
<213> Unknown (Unknown)
<400> 29
gaacgacatg gctacgatcc gacttctcag tgagaagcgc acgct 45
<210> 30
<211> 54
<212> DNA
<213> Unknown (Unknown)
<400> 30
tcctaagacc gcttggcctc cgacttnnnn nngcagcatc tcatcctgaa gttg 54
Claims (22)
1. A method for detecting organism fusion gene and quantifying fusion abundance, which takes organism nucleic acid as a template of reverse transcription and amplification reaction, namely a reaction template, uses a reverse transcription primer extension strategy to carry out reverse transcription and amplification on the template, then carries out organism fusion gene detection and quantifies fusion abundance, and is characterized in that: the organism fusion gene detection comprises the steps that at least one reverse transcription primer carries out reverse transcription reaction under the action of reverse transcriptase; amplifying by using 5 ' end specific forward primers of various heterologous upstream partner genes, and any two primers of a3 ' end specific gene reverse primer containing a kinase domain coding region, a sequencing joint in a Tn5 transposition complex, a forward primer at one end of a sequencing platform specificity or a 5 ' end of a reverse primer with the other end, wherein the amplification is single primer extension, isothermal amplification or PCR amplification; introducing a specific sequence at the 5' end of the template-dependent forward primer or the template-dependent reverse primer; introducing a specific sequence into Tn5 transposition complex; or a specific sequence is introduced into the 5' end of the forward primer at one specific end of the sequencing platform or the reverse primer with the other end; the specific sequence is a molecular marker, and the molecular marker comprises a molecular label; displaying, calculating and quantifying the abundance of the fusion transcript by using a molecular marker method; presentation of fusion results and quantification of fusion transcript abundance were performed using molecular biology research methods of second generation sequencing, third generation sequencing, fluorescent quantitative PCR, digital PCR, gene chip, mass spectrometry, capillary electrophoresis or Sanger sequencing.
2. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the reaction template is nucleic acid of the organism.
3. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the reverse transcriptase has RNA-dependent DNA polymerase activity, DNA-dependent DNA polymerase activity and terminal nucleotidyl transferase activity, the terminal transferase activity is only shown when the reverse transcriptase contacts the 5 'end of an RNA template, additional nucleotides are added to the end of cDNA 3', the length of the added nucleotide sequence is 1-3nt, the sequence is any one of Poly (A), Poly (G), Poly (C) and Poly (T), and the additional added nucleic acid is also called template-free dangling; the cDNA3 'end is added with additional nucleotides to complementarily match with complementary bases, such as a primer containing any one of Poly (A), Poly (G), Poly (C) and Poly (T) at its 3' end, and 1-3nt in length, which can initiate template displacement reaction, such primer containing PCR primer anchor site (i.e., template displacement primer) to perform PCR amplification in combination with reverse transcription primer. In addition, if a single terminal transferase is used after reverse transcription is finished, more template-free overhangs with the length of 1-300nt can be added, and the template-free overhangs can also be complementarily paired with the template displacement primers, so that PCR amplification can be carried out in combination with the reverse transcription primers.
4. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the reverse transcription primer is one, any two combinations or three of oligo d (T), random primer or single-ended template-dependent primer generated single-strand cDNA; the primer for template replacement is a primer containing a molecular label and common use of a sequencing platform, the 3' end is a base which is vertically complementary to the template-free overhang at the end of a strand, and the length of the primer is 1-50nt, wherein the base is at least one of Poly (A), Poly (G), Poly (C) and Poly (T); the reverse transcription primer (or a combination thereof) may be combined with a template displacement primer to perform amplification.
5. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: designing a template-dependent forward primer by the upstream chaperone gene 10-500nt away from the fusion breakpoint and 5'; the 3' end contains the gene design template dependent reverse primer of the kinase domain coding region, and the primer length is 15-50 nt.
6. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the molecular marker is a sequence with specific length, the length of the molecular marker is 2-200nt, and the sequence is a random base N (A/G/C/T), or a semi-random base M (A/C), V (A/C/G), R (A/G), H (A/C/T), W (A/T), D (A/G/T), S (C/G), B (C/G/T), Y (C/T), K (G/T), or a combination of a random base and a fixed base, or a combination of a semi-random base and a random base.
7. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the molecular marker is added to a sequencing joint in a template-dependent forward primer, a template-dependent reverse primer, a Tn5 transposition complex, a forward primer at one specific end of a sequencing platform or a reverse primer with the other end, or is added to any two amplification primers.
8. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 2, wherein the method comprises the following steps: the source of the organism is one or more than one of fresh tissue, fixed tissue, FFPE slices, paraffin rolls, paraffin sample blocks, blood, plasma, serum, saliva, body fluid, urine, milk, follicular fluid and semen, and the nucleic acid template is nucleic acid of the organism, including Total RNA, mRNA, miRNA, long-chain non-coding RNA or DNA.
9. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the template amplification is directly carried out by taking DNA as a substrate, or Total RNA as a substrate, or mRNA, miRNA and lncRNA as substrates, preferably Total RNA as a substrate, and can be single-stranded RNA, RNA/DNA hybrid strand or double-stranded cDNA.
10. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 9, wherein the method comprises the following steps: before amplification is carried out by taking Total RNA as a substrate or mRNA as a substrate, reverse transcription or in vitro transcription is required, and the reverse transcription is preferred.
11. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the cDNA generated by reverse transcription is single-stranded or double-stranded; the cDNA length is 20-100000 nt.
12. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the template amplification is one of single primer extension, isothermal amplification, multiple displacement amplification or PCR amplification.
13. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the product of the reaction template after amplification can be used as fluorescent quantitative PCR or digital PCR.
14. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the product of the reaction template after amplification is a second generation sequencing library and a third generation sequencing library.
15. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the product of the amplified reaction template is the pre-machine product of a gene chip, mass spectrometry, capillary electrophoresis or Sanger sequencing method.
16. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the organism is an organism with reference genome information, preferably a human or a mouse.
17. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the organism fusion gene detection is used for detecting tumor fusion genes and identifying the abundance of fusion transcripts; the tumors include solid tumors and hematological tumors.
18. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 17, wherein the method comprises the following steps: the tumor fusion gene detection is performed on human tumor fusion genes and comprises RNA extraction, reverse transcription, amplification of an amplification primer, purification and addition of an NGS library building joint.
19. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 1, wherein the method comprises the following steps: the fusion reverse transcription reaction converts RNA into cDNA through reverse transcription, PCR amplification is carried out on a target sequence by using a specific primer with a molecular tag, two rounds of PCR enrichment amplification are carried out on an amplification product after purification and enrichment, and finally a sequencing library is obtained through purification.
20. The method for detecting fusion genes and quantifying fusion abundance of an organism according to claim 19, wherein the method comprises the following steps: the RNA reverse transcription and PCR amplification reaction is completed in a single tube.
21. A method and a kit for detecting organism fusion genes and quantifying fusion abundance are characterized in that: the detection process comprises the following steps:
1) extraction of RNA: extracting RNA from a tissue or blood sample using an RNA extraction kit;
2) reverse transcription and amplification of specific fragments: reverse transcription and a first round of PCR (amplification of a specific target fragment) reaction are completed in a single tube, RNA is taken as a template, a fused 3' end gene reverse primer with a molecular tag is taken as a reverse transcription specific primer, and cDNA is obtained; amplifying the cDNA by using a specific multiplex PCR primer and an amplification reagent to obtain a specific target fragment;
3) and (3) purifying a product: adding quantitative magnetic beads into the PCR amplification product obtained in the last step for purification, and removing RNA and residual amplification primers;
4) connecting a sequencing joint: adding a specific label and a joint reagent in an NGS library establishing reagent into the purified product obtained in the previous step, and performing PCR amplification to obtain a library fragment;
5) library detection: quantifying the library prepared in the last step by using a Qubit, performing quality inspection by using an Agilent Bioanalyzer 2100 or an instrument with the same function, and detecting the concentration of the library and the fragment size of the library to ensure that the final library fragment is in the range of 220-250 bp;
6) and (3) machine sequencing: cyclizing the library qualified in the previous step, and performing high-throughput sequencing by using a sequencing platform;
7) and (4) analyzing the data result by using bioinformatics analysis software.
22. The method and kit for detecting fusion genes and quantifying fusion abundance of an organism according to claim 21, wherein the method comprises the following steps: the bioinformatics analysis software comprises a computer medium of data, comprising the following analysis steps, in particular:
1) according to Q30, firstly, the sequencing quality of the base is evaluated, and simultaneously, the molecular tag sequence in the sequencing sequence is subjected to Mapping and counting; duplification determination based on the diversity of the molecular tag sequences (sequences with the same tag are considered to be derived from the same initial RNA template, which is amplified during PCR);
firstly, comparing the Reads with a reference genome or transcriptome, removing a repeated molecular tag, screening out the number of the Reads which accords with the corresponding Split Reads (the Reads containing a breakpoint of two gene fusions) and the number of wild-type Reads of the 3' end gene of the trans-intron region, and calculating: n (a)Split Reads)/[(N(Split Reads)+N(wild type Reads)]And further obtaining the fusion ratio of the RNA in the original state.
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