WO2020191521A1 - Séquence nucléotidique, et procédé de construction d'une banque de séquençage de zone cible d'arn et application correspondante - Google Patents

Séquence nucléotidique, et procédé de construction d'une banque de séquençage de zone cible d'arn et application correspondante Download PDF

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WO2020191521A1
WO2020191521A1 PCT/CN2019/079235 CN2019079235W WO2020191521A1 WO 2020191521 A1 WO2020191521 A1 WO 2020191521A1 CN 2019079235 W CN2019079235 W CN 2019079235W WO 2020191521 A1 WO2020191521 A1 WO 2020191521A1
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sequence
primer
sequencing
universal
rna
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Chinese (zh)
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杨林
夏军
张艳艳
吕硕
陈芳
梅智颖
陈大洋
蒋慧
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深圳华大智造科技有限公司
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Priority to CN201980091855.0A priority Critical patent/CN113557300A/zh
Priority to PCT/CN2019/079235 priority patent/WO2020191521A1/fr
Publication of WO2020191521A1 publication Critical patent/WO2020191521A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6809—Methods for determination or identification of nucleic acids involving differential detection
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
    • C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms

Definitions

  • the invention relates to the field of gene detection, in particular to a construction method and application of a nucleic acid sequence and RNA target region sequencing library.
  • RNA sequencing (RNAseq) technology has been widely used in gene expression research, and it can sample expressed genes without bias. Compared with quantitative PCR, it can analyze a wider range of genes simultaneously, and the accuracy of RNAseq sequencing results is higher than that of the chip method.
  • the expression profile of eukaryotic cells is characterized by a large number of transcripts, a complex variable splicing situation, and large differences in expression levels.
  • RNAseq sequencing results are scattered throughout the genome, and the sequencing depth of different genes is significantly different, and the sequencing depth of transcripts with low transcription level is often insufficient, which hinders the splicing and quantitative research of variable splicing of specific genes.
  • BCR/ABL1 Since the first fusion gene (BCR/ABL1) was tested and confirmed in the early 1980s, the fusion gene has become an important marker for multiple cancers, for example: BCR/ABL1 is used to detect chronic myelogenous leukemia; EML4/ALK is used Detection of malignant lung adenocarcinoma; TMPRSS2/ERG is used to detect prostate cancer, etc. Since the beginning of the 21st century, with the rise of targeted drugs, cancer treatment drugs, especially non-small cell lung cancer (NSCLC) drugs, have also begun to use the metabolic pathways where fusion genes are located.
  • NSCLC non-small cell lung cancer
  • crizotinib was approved by the FDA for the treatment of ALK and ROS1 gene fusion
  • alectinib was approved by the FDA for the treatment of ALK gene fusion
  • sorafenib and sunitinib received RET gene Converged treatment license.
  • fusion genes The early detection of fusion genes was based on traditional tissue biopsy, using fluorescence in situ hybridization, chromosome band analysis, and RT-PCR analysis techniques to confirm whether the patient’s tumor cells had a specific gene fusion.
  • these early cytology and molecular biology techniques have the following shortcomings for the detection of fusion genes: 1) Long detection time; 2) High false positive rate; 3) High requirements for detection technology, requiring skilled and experienced detection personnel. Conducive to standardization; 4) When the content of fusion gene genotype cells is low in cancer tissue, it cannot be effectively detected; 5) Multiple fusion genes cannot be detected.
  • RNAseq With the development of high-throughput technology, more and more people use RNAseq to detect fusion genes. However, due to the large amount of eukaryotic transcript data, RNAseq of full transcripts causes a lot of unnecessary waste, so the fusion gene Detection needs to be further improved.
  • an object of the present invention is to provide a nucleic acid sequence and a method and application for constructing a sequencing library for RNA target regions using this nucleic acid sequence.
  • target region capture technology Using target region capture technology, the gene or genome segment of interest can be enriched, so that targeted sequencing can be achieved.
  • RNAseq that is, the RNAseq sequencing library is first subjected to sequence capture experiments to enrich the transcripts of interest, and then sequenced to achieve targeted RNAseq or RNA capture sequencing (CaptureSeq).
  • CaptureSeq RNA capture sequencing
  • This method can increase the sequencing depth of target transcripts, perform sensitive gene discovery, efficient transcript assembly and accurate gene expression quantification.
  • This technology combines the application of target area capture technology and RNAseq technology, which can be called RNA capture sequencing technology. Use RNA capture sequencing technology to study the expression of specific genes in samples.
  • This method not only has the advantages of RNASeq compared to other expression profiling research methods, but also allows for purposeful, multi-sample research. Especially in the study of transcripts with low expression levels, it is possible to better splice full-length transcripts, discover new genes, and quantitatively analyze them.
  • the present invention uses a nucleic acid sequence with a 5'end with a universal sequencing sequence and a 6-10 nt random primer sequence at the 3'end to reverse transcribe an RNA sample to obtain a cDNA strand, and then perform reverse transcription on the obtained cDNA
  • the chain is subjected to nested PCR multiplex amplification, and the obtained multiplex PCR product has a complete sequencing adapter, which can be directly subjected to high-throughput sequencing.
  • these RNA samples can be total RNA, mRNA, and cfRNA (plasma free DNA) in biological samples.
  • the present invention provides the following technical solutions:
  • the present invention provides a nucleic acid sequence, comprising: a first general sequencing sequence and a random sequence, the first general sequencing sequence is connected to the random primer sequence, and the first general sequencing sequence It is located at the 5'end of the nucleic acid sequence, and the random primer sequence is located at the 3'end of the nucleic acid sequence, wherein the random primer sequence contains 6-10 random nucleotides.
  • the nucleic acid sequence provided by the present invention includes at least two parts, one is the first universal sequence for sequencing at the 5'end, and the other is the random primer sequence at the 3'end.
  • the first universal sequence for sequencing and the random primer sequence can pass 3'-5 'The phosphodiester bond is directly connected.
  • the first sequencing universal sequence can be any sequencing adapter sequence (partial or complete sequence) of any sequencing platform, or any known fixed sequence, which is used in the subsequent PCR amplification process.
  • the random primer sequence contains 6-10 random nucleotides, for example, 8 random nucleotides. Random nucleotide means that the base on the nucleotide can be any of A, T, C, and G.
  • the random primer sequence is used to use the RNA sample as a template for binding with the RNA sample.
  • the nucleic acid sequence provided by the present invention can be used as a primer for a reverse transcription reaction, that is, a cDNA strand is obtained by reverse transcription using an RNA sample as a template, and the obtained product is used for subsequent PCR amplification.
  • nucleic acid sequence may further include the following additional technical features:
  • the random primer sequence contains 8 random nucleotides.
  • the nucleic acid sequence further comprises: a first molecular tag sequence, the first molecular tag sequence is located between the first sequencing universal sequence and the random primer sequence; the complementary sequence, the The complementary sequence is located between the first molecular tag sequence and the random primer sequence, and the complementary sequence is complementary to a partial sequence of the first sequencing universal sequence.
  • a molecular tag sequence is added between the first sequencing universal sequence and the random primer sequence, the number of original RNA templates is counted through a large number of different molecular tag sequences, and the number of original templates can be traced through subsequent statistics on molecular tags. Number, which can realize the quantitative study of RNA template.
  • the molecular tag sequence can be "wrapped", that is, a complementary sequence is inserted between the random primer sequence and the molecular tag sequence.
  • the partial sequence at the 5'end of the sequencing universal sequence is reverse complementary.
  • the sequence at the 5'end of the nucleic acid sequence can anneal to the complementary sequence to form a neck loop structure, thereby ensuring that the molecular tag is wrapped inside and preventing non-specific binding or amplification with the random primer sequence.
  • the first molecular tag sequence contains 8-20 random nucleotides.
  • the first molecular tag sequence contains 8-20 bp random bases, which can produce 4 8-20 different molecular tag sequences. Since the number of RNA templates is certain at the beginning, when the molecular tag sequence is much larger than the number of RNA templates, each RNA A unique molecular tag is added to the template. The number of original RNA templates is marked by a large number of different molecular tags, and the number of original templates can be traced through subsequent statistics on the types of molecular tags.
  • the complementary sequence is complementary to a partial sequence at the 5'end of the first sequencing universal sequence.
  • the present invention provides a method for constructing a sequencing library of an RNA target region, which includes: using a reverse transcription primer and a reverse transcriptase to perform a reverse transcription reaction based on an RNA sample to obtain the first strand of cDNA,
  • the reverse transcription primer is the nucleic acid sequence according to any one of the embodiments of the first aspect of the present invention
  • the first round of PCR amplification is performed using the upstream specific primer and the first universal primer
  • a first amplification product is obtained, the first universal primer overlaps with a partial sequence of the first sequencing universal sequence, and the binding site of the upstream specific primer is located upstream of the target region; based on the first amplification
  • use downstream specific primers, second universal primers and tag primers to perform a second round of Nested PCR amplification to obtain the target region sequencing library.
  • the 5'end of the downstream specific primers contains part or all of the second Universal sequencing sequence
  • the 5'end of the second universal primer contains a phosphate group, part or all of the sequence of the second universal primer and the downstream specific primer overlap
  • the binding site of the downstream specific primer is located Upstream of the target region
  • the tag primer overlaps with a partial sequence of the first universal primer
  • the binding site of the 3'end of the downstream specific primer is located downstream of the upstream specific primer.
  • the random primer sequence on the nucleic acid sequence can be randomly anchored on all RNA molecules to synthesize a strand of cDNA, and then the target region can be enriched by the general sequencing sequence, upstream specific primer and downstream specific primer. It is expressed as: using multiple upstream specific primers and the first universal primer, using one strand of cDNA as a template to perform the first round of PCR amplification, using multiple downstream specific primers, second universal primers and tag primers for the second
  • the round-nested PCR amplification can capture multiple target regions and analyze the target regions through high-throughput sequencing technology to complete the detection and analysis of multiple genes at one time. This method can use as much cDNA template as possible, thereby improving the utilization efficiency of original RNA molecules. Moreover, this method can detect fusion genes, as well as the fusion of unknown genes.
  • the construction method described above may further add the following technical features:
  • the RNA is cfRNA (plasma free DNA), highly degraded RNA, or RNA with a length of 200-300 bp.
  • the RNA with a length of 200-300 bp is obtained by incubating a long-segment RNA sample with Mg 2+ .
  • Mg 2+ incubation can be used to achieve RNA fragmentation, which can be used to construct a sequencing library for RNA target regions.
  • the tag primer contains a second molecular tag sequence for distinguishing different samples.
  • the 3'end of the tag primer and the 5'end of the first universal primer overlap (or can also be referred to as coincidence).
  • coincidence When two nucleic acid sequences corresponding to nucleic acids overlap, it means that two nucleic acids
  • the sequence corresponds to the same nucleic acid base), and there is a molecular tag sequence in the middle of the tag primer (in order to distinguish it from the first molecular tag sequence in the aforementioned nucleic acid sequence, it is referred to herein as the second molecular tag sequence).
  • the second molecular tag sequence is 8-12 nucleotides. These molecular tag sequences are fixed sequences on some sequencing platforms, and each sequencing platform has its own set of molecular tag sequences, such as barcode sequences or index sequences, and the length of these molecular tag sequences can be 10 nucleotides.
  • the construction method further includes: in the process of designing the upstream specific primer or the downstream specific primer, the design may be performed on at least one intron of the genome.
  • the primers are used in the same way as the RNA samples are amplified, and are used as quality control primers to check whether the RNA samples are contaminated by DNA during the amplification process.
  • These upstream specific primers or downstream specific primers used as quality inspection primers can be designed for target regions or non-target regions.
  • the present invention provides a method for detecting target gene mutations, including:
  • sequencing Based on the target region library, sequencing to obtain sequencing data
  • the sequencing data is compared with the reference genome to determine the mutation result of the target gene.
  • the reference genome is the human genome hg19.
  • the mutation includes at least one of gene fusion, SNV, gene deletion, and gene insertion mutation.
  • the mutation is gene fusion.
  • the gene fusion is EML4-ALK gene fusion.
  • the present invention provides an EML4-ALK fusion gene detection method, which includes: based on an RNA sample, a reverse transcription reaction is performed using a reverse transcription primer and a reverse transcriptase to obtain the first strand of cDNA,
  • the reverse transcription primer is one of SEQ ID NO: 1 or SEQ ID NO: 2; based on the first strand of the cDNA, the first round of PCR amplification is performed using the upstream specific primer and the first universal primer ,
  • the first amplification product is obtained, the first universal primer is SEQ ID NO: 3, and the upstream specific primer is at least one selected from SEQ ID NO: 6 to SEQ ID NO: 30; based on the For the first amplification product, perform a second round of Nested PCR amplification using downstream specific primers, second universal primers and tag primers to obtain the target region sequencing library, and the second universal primer is SEQ ID NO: 4,
  • the tag primer is SEQ ID NO: 5 and the downstream specific primer is at least one of SEQ
  • Figure 1 is a schematic diagram of the structure of a nucleic acid sequence provided according to an embodiment of the present invention.
  • Fig. 2 is a schematic structural diagram of a nucleic acid sequence with a molecular tag sequence and a complementary sequence provided according to an embodiment of the present invention.
  • Fig. 3 is an experimental flowchart of a method for constructing an RNA target region sequencing library by using some nucleic acid sequences according to an embodiment of the present invention.
  • Fig. 4 is an experimental flow chart of a method for constructing an RNA target region sequencing library using some nucleic acid sequences according to an embodiment of the present invention.
  • Fig. 5 is an experimental flow chart of constructing a sequencing library for highly degraded RNA or cfRNA according to an embodiment of the present invention.
  • Fig. 6 is a schematic diagram of primer design for fusion gene detection provided according to an embodiment of the present invention.
  • upstream specific primer refers to a specific primer whose binding site is located upstream of the target region.
  • downstream specific primer also refers to a specific primer whose binding site is located upstream of the target region, but compared to an upstream specific primer, the binding site of a downstream specific primer is located downstream of an upstream specific primer.
  • the binding site of the downstream specific primer is located downstream of the upstream specific primer, not only includes: all the binding sites of the downstream specific primer are located downstream of the upstream specific primer, but also includes: downstream specific In the case where the partial binding site of the sexual primer overlaps with the partial binding site of the upstream specific primer, as long as the last binding site of the downstream specific primer is located downstream of the last binding site of the upstream specific primer.
  • RNA capture sequencing technology has natural advantages. It can purposefully capture RNA fragments of interest for sequencing analysis, providing a basis for disease diagnosis.
  • the analysis steps after sequencing include alignment, assembly, removal of non-target sequences, discovery of new genes (exons), and quantification.
  • the present invention provides a nucleic acid sequence.
  • the nucleic acid sequence provided by the present invention is a conventional primer structure, and the nucleic acid sequence includes a 6-10 nt random primer sequence at the 3'end and the first general sequencing sequence at the 5'end, as shown in FIG. 1.
  • the random primer sequence at the 3'end can be used for random complementary pairing with RNA, so that the extracted total RNA or mRNA is used as a template to realize the synthesis of the first strand of cDNA.
  • the length of the random sequence at the 3'end is 6-10 nt, preferably 8 nt.
  • the 5'end first sequencing universal sequence can be any sequencing adapter sequence (partial or complete sequence) of any sequencing platform, or any fixed sequence. Using this nucleic acid sequence, qualitative research on RNA samples can be performed.
  • the present invention also provides another nucleic acid sequence.
  • the nucleic acid sequence is different from the above-mentioned nucleic acid sequence in that in addition to the first sequencing universal sequence and the random primer sequence, it also has a first molecular tag sequence.
  • complementary sequence as shown in Figure 2 (in order to make it easier to observe the 5'end of the first sequencing universal sequence can be reverse-complemented with the complementary sequence or can also be called reverse-paired sequence, the segment can be paired with the complementary sequence
  • the sequence of is shown with a different line from the first sequencing general sequence, and the same strategy is used in Figure 3).
  • the 3'end of the nucleic acid sequence is a 6-10 nt random primer sequence, and the 5'end has a 8-20 nt random first molecular tag sequence in addition to the first sequencing general sequence.
  • the molecular tag sequence is "wrapped" in the design, and a complementary sequence is inserted between the random primer sequence and the molecular tag sequence.
  • the complementary sequence and 5 The partial sequence of the first sequencing universal sequence at the end is reverse complementary. Under normal circumstances, the 5'end sequence and the complementary sequence can anneal to form a neck loop structure to ensure that the molecular tag is wrapped inside to prevent non-specific binding or amplification.
  • the nucleic acid sequence can be used for qualitative and accurate quantitative research on RNA samples.
  • the present invention also provides a method for constructing an RNA target region sequencing library, which includes: based on an RNA sample, a reverse transcription reaction is performed using a reverse transcription primer and a reverse transcriptase to obtain the first strand of cDNA, wherein the reverse transcription primer is The nucleic acid sequence according to any one of the embodiments of the first aspect of the present invention; based on the first strand of the cDNA, the first round of PCR amplification is performed using the upstream specific primer and the first universal primer to obtain the first amplification product, The first universal primer overlaps a partial sequence of the sequencing universal sequence, and the binding site of the upstream specific primer is located upstream of the target region; based on the first amplification product, the downstream specific primer, The tag primer and the second universal primer are subjected to the second round of Nested PCR amplification to obtain the target region sequencing library, the 5'end of the second universal primer contains a phosphate group, the second universal primer and the downstream The partial sequence of the specific primer overlaps, the tag
  • RNA target region sequencing library For ease of understanding, referring to FIG. 3, FIG. 4 or FIG. 5, the method for constructing the above-mentioned RNA target region sequencing library will be described.
  • RNA fragmentation is not required for fragmented RNA or highly degraded RNA such as cfRNA.
  • RNA can be used as a template and incubated for 30 minutes under the action of reverse transcription primers and reverse transcriptase to obtain the first strand of cDNA, and the obtained first strand is purified to remove excess primers and ions.
  • the first strand of cDNA refers to the strand that is complementary to RNA synthesized by reverse transcription using RNA as a template.
  • the reverse transcription primer is the nucleic acid sequence mentioned above.
  • the upstream specific primer is the target region to be studied, such as the target gene or the gene sequence of the upstream region of the target site.
  • the upstream specific primer should be on EML4; the first universal primer and the reverse transcription primer have the same partial sequence of the first sequencing universal sequence at the 5'end, and the product from the first round of PCR With upstream specific primer sequence and first universal sequence.
  • the amplified product is purified to remove excess upstream specific primers and ions.
  • downstream specific primers consist of a partial sequencing adapter sequence at the 5'end (this partial sequencing adapter sequence is part of the sequence of the second universal primer). Overlap or coincide) and gene-specific sequence composition, the gene-specific sequence is downstream of the upstream specific primer, and the two can overlap or be separated.
  • the upstream and downstream specific primers are all set on the upstream gene (such as EML4-ALK, the upstream and downstream specific primer sequences should be on EML4).
  • tag primers are used to distinguish different samples. After the sequencing library is built, equal amounts can be mixed for on-machine sequencing.
  • the 5'end of the second universal primer contains a phosphate group, and the second universal primer can make the 5'end of the product obtained through the second round of amplification have a phosphate group, thereby avoiding the excessively high cost of phosphorylation.
  • the binding sites of the downstream specific primers are located downstream of the upstream specific primers. While the amplification is being carried out, there are two rounds of specific screening, making the amplification results more specific.
  • quality inspection primers can be designed for quality inspection.
  • the quality inspection primers are designed across exons and introns or on introns. As shown in Figure 6. Under normal circumstances, DNA quality control primers cannot amplify products. When DNA is contaminated, products can be amplified.
  • RNA is sequenced, and then the RNA information can be obtained by comparing with the reference genome.
  • RNA is reverse transcribed with reverse transcription primers with random primer sequences.
  • the cDNA after reverse transcription is directly used for nested PCR, which requires a small template length and low primer footprint, which greatly improves the utilization efficiency of the template.
  • two rounds of PCR amplification are carried out with a unidirectional nested primer combined with a universal primer.
  • One end of the amplified product is a fixed end and one end is a random end. This solution can perform any unknown fusion mutations in the target gene and target position.
  • Detection For example, for unknown fusions, for example, to study all fusions occurring on EML1 exon, then design upstream and downstream specific primers on EML1 exon. Because the present invention amplifies RNA samples in a fixed sequence at one end, The sequence bound by the primer at one end is added to the first universal sequence for sequencing randomly anchored to the RNA, so any random fusion with EML1 exon can be detected.
  • the first molecular tag sequence is used to label the reverse transcribed cDNA, which can accurately count the absolute amount of the original mRNA molecule, and the mutation can be corrected through the molecular tag combined information analysis algorithm to improve the detection of RNA mutations accuracy.
  • the entire RNA target library construction experiment process has only three steps, reverse transcription, the first round of PCR, and the second round of PCR. Compared with conventional RNAseq and probe hybridization-based capture technology, it greatly simplifies the experimental process. .
  • Primer design A total of 20 pairs of upstream and downstream primers are designed on the exon of EML4, and a pair of upstream and downstream internal reference primers are designed on the intron.
  • the primer sequence is shown in Table 2, forming an EML4-ALK detection panel, which can be paired Test all possible fusion genes on EML4 gene;
  • *N is a random base
  • #N is a second molecular tag sequence
  • this second molecular tag sequence may also be called a sample tag sequence.
  • N is the base A, T, C or G.
  • the upstream primer pool is composed of 10 ⁇ M of the above primers and other concentrations.
  • the number in Table 2 represents: Take EML4-E02-01-USP as an example, where EML4 represents the gene, E02 represents the second exon, 01 represents the first pair of primers of the first exon, and USP represents the upstream specificity Sexual primers, etc.
  • DSP in Table 3 represents downstream specific primers.
  • the downstream primer pool is mixed with 10 ⁇ M of the above primers and other concentrations.
  • the number of each downstream specific primer has the same meaning as the number of the upstream specific primer.
  • the determination includes the following steps:
  • the obtained product was purified by adding 30ul XPure beads (beackman's agecourt AMPure XP magnetic beads, catalog number A63881), and the obtained product was dissolved in 20ulTE.
  • the analysis steps include alignment, splicing, and removal of non-target sequences
  • positive sample 1 to positive sample 4 respectively represent positive samples from the same person; negative sample 1 to negative sample 4 represent negative samples from the same person.
  • EML4 transcribed mRNA EML4 transcribed mRNA
  • EML4-ALK abnormally transcribed mRNA
  • EML-ALK fusion was not detected in the negative sample, and the target gene fusion (EML-E13-ALK/EML-E14-ALK) was detected in the positive sample with two primers, and the fusion ratio was EML respectively.
  • the method of the present invention can detect gene fusion.
  • first and second are only used for descriptive purposes, and cannot be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. Therefore, the features defined with “first” and “second” may explicitly or implicitly include at least one of the features.
  • “plurality” means at least two, such as two, three, etc., unless otherwise specifically defined.

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Abstract

L'invention concerne une séquence nucléotidique, et un procédé de construction d'une bibliothèque de séquençage de zone cible d'ARN et une application correspondante. La séquence nucléotidique comprend une première séquence de séquençage générale située à l'extrémité 5' et une séquence d'amorce aléatoire située au niveau de l'extrémité 3' étant reliées l'une à l'autre. La séquence d'amorce aléatoire contient de 6 à 10 nucléotides aléatoires. La séquence est utilisée pour effectuer une transcription inverse aléatoire sur l'ARN pour obtenir une première chaîne d'ADNc, puis la première séquence de séquençage générale sur la séquence de nucléotides et des amorces spécifiques amont et aval sont utilisées pour effectuer une PCR nichée en deux étapes sur la première chaîne d'ADNc obtenue après la transcription inverse, pour obtenir une banque de séquençage ciblée à haut débit.
PCT/CN2019/079235 2019-03-22 2019-03-22 Séquence nucléotidique, et procédé de construction d'une banque de séquençage de zone cible d'arn et application correspondante WO2020191521A1 (fr)

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