WO2024001404A1 - 检测脆性x综合征突变的方法和试剂盒 - Google Patents

检测脆性x综合征突变的方法和试剂盒 Download PDF

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WO2024001404A1
WO2024001404A1 PCT/CN2023/087226 CN2023087226W WO2024001404A1 WO 2024001404 A1 WO2024001404 A1 WO 2024001404A1 CN 2023087226 W CN2023087226 W CN 2023087226W WO 2024001404 A1 WO2024001404 A1 WO 2024001404A1
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fmr1
pcr amplification
fxs
sequencing
cgg
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French (fr)
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孟万利
詹嘉晗
毛爱平
李佳琪
卢玉林
张丽
任志林
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北京贝瑞和康生物技术有限公司
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    • 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
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    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
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    • 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
    • C12Q1/6869—Methods for sequencing
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • the invention relates to a primer and method for detecting multiple mutations of FXS using a third-generation long-read sequencing platform, as well as a kit suitable for this method.
  • Fragile X syndrome is an X-linked incomplete penetrance genetic disease and one of the common chromosomal diseases. It is named after the brittle part that appears to be broken.
  • FXS is the most common cause of inherited intellectual disability, affecting 1/4000 males and 1/8000-1/4000 females. In most cases, FXS is caused by a trinucleotide (CGG) repeat expansion in the 5'-UTR region of the FMR1 gene on the X chromosome (Crawford D C et al., Genetics In Medicine, 2001, 3(5):359-371 ).
  • CGG trinucleotide
  • Trinucleotide can be divided into premutation (55-200) and complete mutation (>200) according to the number of repeats; premutation is unmethylated and can produce FMR1 protein with slightly lower expression than normal, and is completely High CGG repeats in patients with mutations will be accompanied by hypermethylation, which usually results in transcriptional silencing of the FMR1 gene and loss of FMR1 protein (Kenneson A et al., Hum Mol Genet, 2001; 10:1449–1454; Pieretti M et al., Cell, 1991, 66(4):817-822).
  • FMR1 gene point mutations and large fragment FMR1 gene deletions will also cause symptoms of FXS (Gedeon A K et al., Nature Genetics, 1992, 1(5):341 -344; Handt M et al., MCB, 2014, 28(5-6): 279-283; Collins S C et al., AM J MED GENET A, 2010, 152(10): 2512-2520).
  • the clinical phenotype of FXS is broad and includes intellectual retardation, developmental and language delays, and physical abnormalities; the behavioral phenotype of FXS is also characterized by approximately 25% to 33% of autistic symptoms, including social and communication impairments, and sensitivity to sensory stimulation. Abnormal reactions, etc. (Hagerman RJ et al., Baltimore: Johns Hopkins University Press; 2002.p3–109).
  • the most common method for molecular detection of CGG repeat expansion in FXS is to detect whether it contains more than 200 CGG repeat expansions through polymerase chain reaction (PCR) of capillary electrophoresis (CE). This method can also determine whether there are less than 200 CGG repeat expansions. Number (Filipovic-Sadic S et al., Clinical Chemistry, 2010, 56(3):399-408). However, this method is affected by the detection length and cannot confirm higher CGG repeat numbers; since CE can only infer the CGG repeat number from the length of the PCR product, the accuracy of this method mainly depends on the accuracy of standard selection. Noise in the signal can also have an adverse impact on result judgment (Grasso M et al., JMD, 2014, 16(1):23-31).
  • the Southern blot method of FMR1 is used to detect samples whose CGG repeatability is too high to pass PCR, and is also used to determine the methylation status of the FMR1 gene.
  • the workflow of this method is complicated. The cost is high, requires more time and effort, and requires more sample DNA, so it cannot be adapted to the detection of large batches of samples (Hagerman RJ et al., Baltimore: Johns Hopkins University Press; 2002.p3–109).
  • First-generation sequencing (Sanger) and second-generation sequencing (NGS) cannot detect the CGG repeat expansion in FXS, but they can detect point mutations in the FMR1 gene (Collins S C et al., AM J MED GENET A, 2010,152( 10):2512-2520); Sanger sequencing has low throughput for detecting FMR1 point mutations and cannot detect unknown mutations; while NGS requires the construction of an NGS sequencing library for the FMR1 gene to detect point mutations in the FMR1 gene, and is not compatible with other gene tests. And the process is relatively cumbersome. Multiplex ligation-dependent probe amplification (MLPA) can detect large fragment deletions of the FMR1 gene. However, this method requires simultaneous detection of different upstream and downstream sites of the FMR1 gene, which is cumbersome and cannot detect large quantities of samples ( Hayward B E et al., Human Genetics, 2017, 136(10):1313-1327).
  • MLPA Multiplex ligation-dependent probe amplification
  • CE and Southern blot can only detect CGG repeat numbers greater than 200, and cannot determine higher CGG repeat numbers;
  • CE judgment of the number of CGG repeats less than 200 relies on standards with known results, and it is impossible to directly and accurately determine the number of CGG repeats
  • the present invention provides a method based on long fragment PCR and high GC PCR amplification and third-generation sequencing to detect multiple mutations related to FXS.
  • Long-fragment PCR amplification and high-GC PCR amplification are implemented in one or two reaction tubes; long-fragment PCR amplification is used to detect point mutations and large-fragment deletions of the FMR1 gene; high-GC PCR amplification of the FMR1 gene 5
  • the CGG repeat region of the '-UTR is used to detect premutation and full mutations caused by CGG expansion, AGG insertion in the CGG repeat, and small deletions occurring in the 5'-UTR region.
  • the purpose of this invention is to solve the problems of incomplete detection coverage of FXS pathogenic genes at this stage, the inability to accurately judge CGG repeat expansion and AGG insertion in CGG repeats, and the inability to determine a low proportion of chimeric mutations, which will lead to clinical missed detection and misdetection. .
  • FXS-related pathogenic gene FMR1 and the 5'-UTR region of FMR1 that is prone to CGG repeat expansion and deletion in FXS and prepare a third-generation sequencing library to achieve comprehensive, accurate and rapid Detect multiple mutations in FXS targets across multiple samples.
  • the invention relates to a primer set for multiple mutations of FXS, comprising one or more pairs of primers selected from the following:
  • FMR1-E1-F and FMR1-E1-R wherein said FMR1-E1-F is selected from SEQ ID NO: 1-4, and FMR1-E1-R is selected from SEQ ID NO: 5-8;
  • FMR1-E2-F and FMR1-E9-R wherein said FMR1-E2-F is selected from SEQ ID NO: 9-11, and FMR1-E9-R is selected from SEQ ID NO: 12-14;
  • FMR1-E10-F and FMR1-E17-R wherein said FMR1-E10-F is selected from SEQ ID NO: 15-17, and FMR1-E17-R is selected from SEQ ID NO: 18-20;
  • FMR1-Gap-Mix wherein the FMR1-Gap-Mix is selected from SEQ ID NO: 21-220;
  • the multiple mutations of FXS include at least one or more of the following: Premutation and full mutation of FMR1 caused by CGG expansion, AGG insertion in CGG repeat, large deletion, point mutation and microdeletion.
  • the primer positions are shown in Figure 1A to Figure 1B.
  • the primers can amplify the entire sequence on the genome within the scope of the primer, including any type of mutated sequence within the scope of the primer.
  • the amplification products of each primer are approximately 2.7kb (FMR1-E1), 11.6kb (FMR1-E2-9) and 15.2kb (FMR1-E10-17).
  • degenerate base primers are used if there is a SNP at the primer position.
  • the primer set includes the following pairs of primers:
  • FMR1-E1-F and FMR1-E1-R wherein said FMR1-E1-F is selected from SEQ ID NO: 1-4, and FMR1-E1-R is selected from SEQ ID NO: 5-8;
  • FMR1-E2-F and FMR1-E9-R wherein said FMR1-E2-F is selected from SEQ ID NO: 9-11, and FMR1-E9-R is selected from SEQ ID NO: 12-14;
  • FMR1-E10-F and FMR1-E17-R wherein said FMR1-E10-F is selected from SEQ ID NO: 15-17, and FMR1-E17-R is selected from SEQ ID NO: 18-20; and
  • FMR1-Gap-Mix wherein said FMR1-Gap-Mix is selected from SEQ ID NO: 21-220.
  • the multiple mutations of FXS include at least one or more of the following: pre-mutation and full mutation caused by CGG expansion in the 5'-UTR region of FMR1, AGG insertion and mini-mutation in CGG repeats. Deletions; large deletions and all point mutations within the FMR1-Gap-Mix primer range of FMR1.
  • the primer set of the present invention can simultaneously detect pre-mutation and full mutation of FXS caused by CGG expansion, AGG insertion in CGG repeats, and determine the accurate site of minor deletions in the 5'-UTR region; detect All SNVs of FMR1 and large fragment deletions within the scope of the FMR1-Gap-Mix primer.
  • 5-50 nt DNA of different sequences can be added to the 5' end of the primer to distinguish different samples; preferably, the 5' end of the F and R primers Barcodes can be the same or different, and those skilled in the art can choose according to needs.
  • DNA barcode Barcode
  • the primer set can be used for 1 or 2 systems of PCR amplification of FXS-related pathogenic gene fragments including all ranges of mutation types in the FMR1 gene and the 5'-UTR region of the FMR1 gene. Combined with the subsequent PacBio or Nanopore sequencing platform, the mutation types of the FMR1 gene and all gene fragments within the 5’-UTR region of the FMR1 gene can be detected.
  • a second aspect of the present invention provides the use of the primer set of the first aspect of the present invention in preparing a kit for detecting multiple mutations of FXS, wherein the multiple mutations of FXS include at least one or more of the following: FMR1 Premutation and full mutation caused by CGG expansion, AGG insertion in CGG repeat, large deletion, point mutation and microdeletion.
  • the third aspect of the present invention provides a kit for detecting multiple mutations of FXS, including the following reagents:
  • the reagents for long fragment PCR amplification and high GC PCR amplification include DNA polymerase, reaction buffer and primer set.
  • the primer set in the kit is selected from the following one or more pairs of primers:
  • FMR1-E1-F and FMR1-E1-R wherein said FMR1-E1-F is selected from SEQ ID NO: 1-4, and FMR1-E1-R is selected from SEQ ID NO: 5-8;
  • FMR1-E2-F and FMR1-E9-R wherein said FMR1-E2-F is selected from SEQ ID NO: 9-11, and FMR1-E9-R is selected from SEQ ID NO: 12-14;
  • FMR1-E10-F and FMR1-E17-R wherein said FMR1-E10-F is selected from SEQ ID NO: 15-17, and FMR1-E17-R is selected from SEQ ID NO: 18-20;
  • FMR1-Gap-Mix wherein the FMR1-Gap-Mix is selected from SEQ ID NO: 21-220;
  • the multiple mutations of FXS include at least one or more of the following: premutation and full mutation of FMR1 caused by CGG expansion, AGG insertion in CGG repeats, large fragment deletions, point mutations and microdeletions.
  • the primer positions are shown in Figure 1A to Figure 1B.
  • the primers can amplify the entire sequence on the genome within the scope of the primer, including any type of mutated sequence within the scope of the primer.
  • the amplification products of each primer are approximately 2.7kb (FMR1-E1), 11.6kb (FMR1-E2-9) and 15.2kb (FMR1-E10-17).
  • degenerate base primers are used if there is a SNP at the primer position.
  • the multiple mutations of FXS include at least one or more of the following: pre-mutation and full mutation caused by CGG expansion in the 5'-UTR region of FMR1, AGG insertion and mini-mutation in CGG repeats. Deletions; large deletions and all point mutations within the FMR1-Gap-Mix primer range of FMR1.
  • the primer set of the present invention can simultaneously detect premutation and full mutation of FXS caused by CGG expansion, AGG insertion in CGG repeats, determine the exact site of minor deletions in the 5'-UTR region, and detect All SNVs of FMR1 and large fragment deletions within the scope of the FMR1-Gap-Mix primer.
  • 5-50nt DNA (Barcode) of different sequences can be added to the 5' end of the primers in the kit to distinguish different samples; preferably, the 5' end Barcode of the F and R primers They can be the same or different, and those skilled in the art can choose according to needs.
  • the PCR amplification product can be purified or not purified before proceeding to the next reaction, and those skilled in the art can choose according to needs.
  • reagents for constructing a third-generation sequencing library include end repair enzymes, adapters, ligases, DNA purification magnetic beads, reaction buffers, and exonucleases.
  • long fragment PCR amplification and high GC PCR amplification are completed in one or two reaction tubes.
  • the third-generation sequencing is selected from Pacific Biosciences' PacBio sequencing or ONT's Nanopore sequencing.
  • the reagents used to construct third-generation PacBio sequencing libraries include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol, reaction buffer and exonuclease.
  • PacBio library adapter ligation can use blunt end ligation or TA ligation.
  • the PacBio universal blunt-end adapter sequence is 5’-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAT-3’ (SEQ ID NO: 221), which forms a blunt-end stem-loop structure adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form different adapter adapters with Barcodes.
  • PacBio libraries with different barcodes can be mixed and sequenced together.
  • the PacBio universal TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 222), which forms a blunt-ended stem-loop structure linker adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form different adapter adapters with Barcodes.
  • PacBio libraries with different barcodes can be mixed and sequenced together.
  • the PacBio adapter is available with or without Barcode.
  • the PacBio connector has a Barcode designed by PacBio or a Barcode designed by oneself, and those skilled in the art can choose according to needs.
  • the PacBio library matches the sequencing platform of Pacific Biosciences.
  • the reagents used to construct a third-generation Nanopore library include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.
  • Nanopore library adapter ligation can use blunt end ligation or TA ligation.
  • Nanopore adapters can be provided with or without Barcode.
  • the Nanopore connector has a Barcode designed by ONT Company or a self-designed Barcode. Those skilled in the art can choose according to needs.
  • the Nanopore library matches the sequencing platform of ONT Company.
  • the fourth aspect of the present invention provides a system for detecting multiple mutations of FXS, including the following modules:
  • Collection module obtain subject samples
  • Amplification module perform long fragment PCR amplification and high GC PCR amplification on the sample
  • Sequencing module Sequence and analyze FMR1 gene mutation types.
  • step (2) is performed using the primer set described in the first aspect of the present invention.
  • the multiple mutations of FXS include at least one or more of the following: premutation and full mutation of FMR1 caused by CGG expansion, AGG insertion in CGG repeats, large fragment deletions, point mutations and microdeletions.
  • the primer positions are shown in Figure 1A to Figure 1B.
  • the primers can amplify the entire sequence on the genome within the scope of the primer, including any type of mutated sequence within the scope of the primer.
  • the amplification products of each primer are approximately 2.7kb (FMR1-E1), 11.6kb (FMR1-E2-9) and 15.2kb (FMR1-E10-17).
  • degenerate base primers are used if there is a SNP at the primer position.
  • the long fragment PCR amplification is to amplify FMR1 E2-9, FMR1 E10-17 fragments in the sample.
  • the high GC PCR amplification is amplifying the FMR1 El fragment in the sample.
  • the primer set of the present invention can simultaneously detect premutation and full mutation of FXS caused by CGG expansion, AGG insertion in CGG repeats, determine the exact site of minor deletions in the 5'-UTR region, and detect All SNVs of FMR1 and large fragment deletions within the scope of the FMR1-Gap-Mix primer.
  • 5-50nt DNA (Barcode) of different sequences can be added to the 5' end of the primers in the kit to distinguish different samples; preferably, the 5' end Barcode of the F and R primers They can be the same or different, and those skilled in the art can choose according to needs.
  • long fragment PCR and high GC PCR amplification is completed in one or two reaction tubes.
  • sample is selected from a biological sample or gDNA extracted from a sample.
  • the biological samples are selected from cultured cell lines, blood, amniotic fluid, villi, gametes, blastocyst cells, synovial fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic fluid, etc.
  • the third generation sequencing of the method is selected from Pacific Biosciences' PacBio sequencing or ONT's Nanopore sequencing.
  • the reagents used to construct third-generation PacBio sequencing libraries include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol, reaction buffer and exonuclease.
  • PacBio library adapter ligation can use blunt end ligation or TA ligation.
  • the PacBio universal blunt-end adapter sequence is 5’-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAT-3’ (SEQ ID NO: 221), which forms a blunt-end stem-loop structure adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form different adapter adapters with Barcodes.
  • PacBio libraries with different barcodes can be mixed and sequenced together.
  • the PacBio universal TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 222), which forms a blunt-ended stem-loop structure linker adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form adapter adapters with different Barcodes.
  • PacBio libraries with different Barcodes can be mixed together for sequencing.
  • the PacBio adapter is available with or without Barcode.
  • the PacBio connector carries a Barcode designed by PacBio or a Barcode designed by itself. Those skilled in the art can choose according to needs.
  • the PacBio library matches the sequencing platform of Pacific Biosciences.
  • the reagents used to construct a third-generation Nanopore library include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.
  • Nanopore library adapter ligation can use blunt end ligation or TA ligation.
  • the Nanopore connector can be provided with or without Barcode, and those skilled in the art can choose according to needs.
  • the Nanopore connector has a Barcode designed by ONT Company or a self-designed Barcode. Those skilled in the art can choose according to needs.
  • the Nanopore library matches the sequencing platform of ONT Company.
  • the fifth aspect of the present invention provides a method for detecting multiple mutations of FXS, including the following steps:
  • step (2) is performed using the primer set described in the first aspect of the present invention.
  • the multiple mutations of FXS include at least one or more of the following: premutation and full mutation of FMR1 caused by CGG expansion, AGG insertion in CGG repeats, large fragment deletions, point mutations and microdeletions.
  • the primer positions are shown in Figure 1A to Figure 1B.
  • the primers can amplify the entire sequence on the genome within the scope of the primer, including any type of mutated sequence within the scope of the primer.
  • the amplification products of each primer are approximately 2.7kb (FMR1-E1), 11.6kb (FMR1-E2-9) and 15.2kb (FMR1-E10-17).
  • degenerate base primers are used if there is a SNP at the primer position.
  • the long fragment PCR amplification is to amplify FMR1 E2-9, FMR1 E10-17 fragments in the sample.
  • the high GC PCR amplification is amplifying the FMR1 El fragment in the sample.
  • the primer set of the present invention can simultaneously detect premutation and full mutation of FXS caused by CGG expansion, AGG insertion in CGG repeats, determine the exact site of minor deletions in the 5'-UTR region, and detect All SNVs of FMR1 and large fragment deletions within the scope of the FMR1-Gap-Mix primer.
  • 5-50nt DNA (Barcode) of different sequences can be added to the 5' end of the primers in the kit to distinguish different samples; preferably, the 5' end Barcode of the F and R primers They can be the same or different, and those skilled in the art can choose according to needs.
  • long fragment PCR and high GC PCR amplification is completed in one or two reaction tubes.
  • sample is selected from a biological sample or gDNA extracted from a sample.
  • the biological samples are selected from cultured cell lines, blood, amniotic fluid, villi, gametes, blastocyst cells, synovial fluid, urine, sweat, saliva, feces, cerebrospinal fluid, ascites, pleural effusion, bile or pancreatic fluid, etc.
  • the third generation sequencing of the method is selected from Pacific Biosciences' PacBio sequencing or ONT's Nanopore sequencing.
  • the reagents used to construct third-generation PacBio sequencing libraries include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol, reaction buffer and exonuclease.
  • PacBio library adapter ligation can use blunt end ligation or TA ligation.
  • the PacBio universal blunt-end adapter sequence is 5’-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAT-3’ (SEQ ID NO: 221), which forms a blunt-end stem-loop structure adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form different adapter adapters with Barcodes.
  • PacBio libraries with different barcodes can be mixed and sequenced together.
  • the PacBio universal TA linker sequence is 5'-pATCTCTCTCTTTTCCTCCTCCCGTTGTTGTTGTTGAGAGAGATT-3' (SEQ ID NO: 222), which forms a blunt-ended stem-loop structure linker adapter through annealing.
  • 5-50nt DNA (Barcode) of different sequences can be added to the stem to form adapter adapters with different Barcodes.
  • PacBio libraries with different Barcodes can be mixed together for sequencing.
  • the PacBio adapter is available with or without Barcode.
  • the PacBio connector carries a Barcode designed by PacBio or a Barcode designed by itself. Those skilled in the art can choose according to needs.
  • the PacBio library matches the sequencing platform of Pacific Biosciences.
  • the reagents used to construct a third-generation Nanopore library include end repair enzymes, adapters, ligases, DNA purification magnetic beads, 80% ethanol and reaction buffer.
  • Nanopore library adapter ligation can use blunt end ligation or TA ligation.
  • the Nanopore connector can be provided with or without Barcode, and those skilled in the art can choose according to needs.
  • the Nanopore connector has a Barcode designed by ONT Company or a self-designed Barcode. Those skilled in the art can choose according to needs.
  • the Nanopore library matches the sequencing platform of ONT Company.
  • the method of the present invention based on a specific combination of long fragment PCR and high GC PCR amplification and third-generation high-throughput sequencing can achieve the simultaneous detection of multiple FXS-related pathogenic genes in multiple samples with high specificity, accuracy and speed. kind of mutation.
  • the present invention can simultaneously detect the CGG repeat expansion types of FXS that have been discovered so far, and can detect deletions occurring in the CGG repeat region; it can detect all SNVs of the FMR1 gene and about 2 MB regions upstream and downstream of the FMR1 gene. Large fragments are missing.
  • the detection rate of false detection and missed detection is low:
  • the most common method currently used to detect CGG expansion of FXS is the PCR reaction detection combined with CE.
  • the PCR reaction detection combined with CE.
  • the high GC PCR system of the present invention combined with the third-generation sequencing method can eliminate the influence of noise and more accurately confirm the CGG expansion type in FXS; currently, there is no analysis of the 5'-UTR region of the FMR1 gene
  • the detection of microdeletions and SNVs of the FMR1 gene forms an effective solution. Therefore, some mutations caused by these reasons cannot be detected.
  • the two reaction primer systems of the present invention can detect the above-mentioned mutation types at the same time.
  • the template used for PCR can be peripheral blood, dried blood spots or extracted genomic DNA, or it can be a human cell line or other specific tissue.
  • High-throughput detection Third-generation sequencing can implement 384 types of Barcode connectors, and more Barcode connectors can actually be designed as needed. Or use the dual Barcode system of primer-banded Barcode and adapter-banded Barcode to achieve more Barcode combinations.
  • the high-throughput characteristics of the third-generation sequencing platform determine that high-throughput sample detection can be achieved.
  • PacBio's dumbbell-shaped library can undergo multiple rounds of interpretation during sequencing, and the base accuracy of the sequencing results after correction is greater than 99%. Moreover, PacBio sequencing errors are random, and the base accuracy is greater than 99.9% when corrected through sequencing depth. Therefore, genetic mutations within the detection range of the primers can be accurately interpreted.
  • the Nanopore platform can generate data within minutes, and data analysis can be started within minutes or hours according to actual data volume requirements. When the detection timeliness requirements are relatively high, the Nanopore platform has a time advantage.
  • Figure 1A-1B Schematic diagram of PCR primer design, where Figure 1A shows a schematic diagram of FXS-related FMR1 E1 detection primer design, and Figure 1B shows a schematic diagram of FXS-related FMR1 gene and large fragment deletion detection primer design design.
  • Figure 2A-2B Schematic diagram of gel electrophoresis of FXS amplified fragments, wherein Figure 2A represents a schematic diagram of DNA gel electrophoresis of long fragment PCR amplification of FMR1 E2-9 and FMR1 E10-17, and Figure 2B represents a schematic diagram of high GC PCR amplification of FMR1 E1 Schematic diagram of DNA gel electrophoresis.
  • Figure 3A-3L Representative PacBio sequencing results of FXS-related gene mutation samples.
  • Figure 3A shows CGG expansion in normal human samples (29)
  • Figure 3B shows CGG expansion in premutation carrier samples (29/87)
  • Figure 3C shows Full mutation carrier samples showed CGG expansion (36/439).
  • Figure 3D shows CGG expansion in normal human samples (30).
  • Figure 3E shows CGG expansion in premutation carrier samples (29/118).
  • Figure 3F shows CGG expansion in full mutation samples ( 203/342/725)
  • Figure 3G shows CGG expansion in premutation carrier samples (29/61)
  • Figure 3H shows CGG expansion in full mutation samples (335/659)
  • Figure 3I shows CGG expansion in premutation and full mutation chimeric samples (69/191/221/336)
  • Figure 3J represents the FMR1 point mutation sample
  • Figure 3K represents the FMR1 5'-UTR microdeletion sample
  • Figure 3L represents the FMR1 large fragment deletion sample.
  • Example 1 Amplification and construction of PacBio sequencing library using the PCR method of the present invention
  • Step 1 Long fragment and high GC PCR amplification
  • DNA concentration was determined using Qubit dsDNA HS reagent (ThermoFisher, Cat#Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216). When there are multiple sample PacBio sequencing libraries, equal amounts of libraries can be mixed together to prepare a mixed library.
  • Step 1 Long fragment and high GC PCR amplification
  • DNA concentration was determined using Qubit dsDNA HS Reagent (ThermoFisher, Cat#Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216). When there are multiple sample PacBio sequencing libraries, equal amounts of libraries can be mixed together to prepare a mixed library.
  • Step 1 Long fragment and high GC PCR amplification
  • DNA concentration was determined using Qubit dsDNA HS Reagent (ThermoFisher, Cat#Q32851) on a Qubit 3 Fluoromter (ThermoFisher, Cat#Q33216). When there are multiple sample PacBio sequencing libraries, equal amounts of libraries can be mixed together to prepare a mixed library.
  • Figure 3G shows CGG expansion (29/61) in premutation carrier samples.
  • Figure 3H shows CGG expansion (335/659) in full mutation samples.
  • Figure 3I shows premutation and full mutation mosaicism.
  • the sample CGG expansion (69/191/221/336),
  • Figure 3J represents the FMR1 point mutation sample,
  • Figure 3K represents the FMR1 5'-UTR microdeletion sample, and
  • Figure 3L represents the FMR1 large fragment deletion sample.
  • the peripheral blood genomic DNA of 24 subjects was collected as 24 verification samples.
  • the method (and kit) of the present invention was used to simultaneously detect multiple mutations of FXS-related genes.
  • the PCR-CE method was used to detect the CGG repeat expansion of FXS
  • the Sanger sequencing method was used to detect point mutations and deletions in related genes.
  • the results obtained by using the present invention are compared with the control results. The results are shown in Table 13. The results of the 24 samples are completely consistent.
  • the results detected using the method of the present invention have both specificity and sensitivity reaching 100%.
  • the method of the present invention can directly detect CGG repeat sequences instead of inferring through other methods; therefore, the accuracy of the method of the present invention is better than the PCR-CE method; and among the 24 samples, 7 samples were completely determined by the method of the present invention.
  • the method of the present invention can directly detect all SNVs and large fragment deletions of unknown FMR1, while the PCR-sanger method can only verify known results.

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Abstract

提供一种检测脆性X综合征（FXS）多种突变的引物组、试剂盒和方法。其中所述试剂盒包括以下试剂：（1）用于长片段PCR扩增的试剂；（2）用于高GC区域PCR扩增的试剂；和（3）用于构建三代测序文库的试剂。其中所述方法包括以下步骤：（1）获取受试者样本；（2）对所述样本进行长片段PCR扩增和高GCPCR扩增；（3）构建三代测序文库；（4）测序并分析FMR1基因突变类型。

Description

检测脆性X综合征突变的方法和试剂盒 技术领域
本发明涉及一种利用三代长读长测序平台检测FXS多种突变的引物和方法，以及适用于此方法的试剂盒。
背景技术
脆性X综合征(Fragile X syndrome,FXS)是一种X连锁不完全外显性遗传病，也是常见的染色体病之一，因细胞中X染色体末端在特殊培养基中经诱变剂作用后可显示如同断裂的脆性部位而得名。
FXS是最常见的遗传性智力缺陷的原因，男性的发病率为1/4000，而女性的携带率为1/8000-1/4000。在大多数情况下，FXS是由X染色体上FMR1基因5’-UTR区域三核苷酸(CGG)重复扩张引起的(Crawford D C等，Genetics In Medicine,2001,3(5):359-371)。三核苷酸(CGG)根据重复数可分为前突变(55-200)和完全突变(>200)；前突变是非甲基化的，可以产生表达量略低于正常值的FMR1蛋白，完全突变的患者中高CGG重复将伴随着高甲基化，这通常会造成FMR1基因转录沉默和FMR1蛋白的缺失(Kenneson A等，Hum Mol Genet,2001；10:1449–1454；Pieretti M等，Cell,1991,66(4):817-822)。除此之外，也有报道其他影响FMR1基因表达的突变：如FMR1基因点突变和大片段的FMR1基因缺失也将造成FXS的症状(Gedeon A K等，Nature Genetics,1992,1(5):341-344；Handt M等，MCB,2014,28(5-6):279-283；Collins S C等，AM J MED GENET A,2010,152(10):2512-2520)。FXS临床表型广泛：包括智力发育迟缓，发育和语言迟缓，身体异常；FXS的行为表型还以大约25％至33％的自闭症症状为特征，包括社交和沟通障碍，对感官刺激的异常反应等(Hagerman RJ等，Baltimore:Johns Hopkins University Press；2002.p3–109)。
完全突变的传播只发生在携带完全突变或者前突变的母亲身上，并且母亲携带前突变CGG重复数越高，遗传给后代完全突变的几率越大(Fernandez-Carvajal I等，JMD,2009,11(4):306-310)。大多数携带前突变的人智力正常，但男性容易出现注意力问题、执行功能障碍、社交缺陷和强迫症行为；完全突变的成年男性平均智商(IQ)约为40，因为FXS为X连锁疾病，因此女性通常比男性收到更轻微的影响，完全突变的女性通常智商正常或接近正常，大多数人会有相关的学习障碍和情绪问题(Merenstein S A等，AM J MED GENET,1996,64(2):388-394；Freund L S等，Pediatrics,1993,91(2):321-329；Garber K B等，EJHG,2008,16(6):666-672)。
目前分子检测FXS的CGG重复扩张最常见的方法是通过毛细管电泳法(CE)的聚合酶链式反应(PCR)来检测是否含有大于200的CGG重复扩张，该方法还可以判断小于200的CGG重复数(Filipovic-Sadic S等，Clinical Chemistry,2010,56(3):399-408)。可是该方法受检测长度的影响无法确认更高的CGG重复数；由于CE只能从PCR产物的长度去推断CGG重复数，因此该方法的准确性主要取决于标准品选取的准确性，同时检测信号中的噪音也会对结果判断造成不利的影响(Grasso M等，JMD,2014,16(1):23-31)。除此之外，FMR1的DNA印迹杂交法(Southern blot)即用于检测CGG重复性太大而无法通过PCR的样品，也用于确定FMR1基因的甲基化状态，可是该方法工作流程复杂，成本高，需要更多的时间和精力，并且需要更多的样品DNA量，因此无法适应于大批量样品的检测(Hagerman RJ等，Baltimore:Johns Hopkins University Press；2002.p3–109)。
一代测序(Sanger)和二代测序(Next-generation sequencing,NGS)无法检测FXS中CGG的重复扩张，但是可以检测FMR1基因的点突变(Collins S C等，AM J MED GENET A,2010,152(10):2512-2520)；Sanger测序检测FMR1点突变通量低，且无法检测未知的突变；而NGS需要对FMR1基因构建NGS测序文库才能检测FMR1基因的点突变，不能与其他基因检测兼容，且流程比较繁琐。多重连接探针扩增技术(multiplex ligation-dependent probe amplification,MLPA)可以检测FMR1基因的大片段缺失，可是该方法需要同时检测FMR1基因上下游不同的位点，操作繁琐且无法检测大批量样本(Hayward B E等，Human Genetics,2017,136(10):1313-1327)。
目前基于CE、Southern blot、Sanger测序或二代测序以及MLPA的方法，可以实现FXS的部分CGG重复扩张检测以及FMR1基因点突变和大片段缺失检测，但检测存在有以下几方面局限性：
1、无法实现在同一体系内同时检测所有的突变类型；
2、由于FXS的CGG重复扩张可能存在一定比例的嵌合体，因此CE和Southern blot可能会漏掉一些低比例的嵌合；
3、CE和Southern blot只能检测大于200的CGG重复数，无法确定更高的CGG重复数数目；
4、CE对于小于200的CGG重复数目的判断依赖于已知结果的标准品，无法直接准确判断CGG重复数；
5、无法准确判断CGG重复中的AGG插入类型；
6、MLPA无法判断大片段缺失的准确位点；
7、目前的方法无法直接检测未知的CGG重复区域附近的微缺失。
发明内容
鉴于此，本发明提供了一种基于长片段PCR和高GC PCR扩增和三代测序的方法来检测FXS相关的多种突变。长片段PCR扩增和高GC PCR扩增被分在一个或者两个反应管中实现；其中长片段PCR扩增用于检测FMR1基因的点突变和大片段缺失；高GC PCR扩增FMR1基因5’-UTR的CGG重复区域用于检测由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入以及5’-UTR区域发生的微小缺失。结合读长测长的三代测序平台读长测长等特点，可以实现准确、快速并高通量地检测FXS相关基因突变。本发明涉及的方法操作简便，长片段PCR以及高GC PCR和三代文库质量可靠且重复性强，有利于三代测序技术在临床检测上的应用。
本发明的目的在于解决现阶段FXS致病基因检测覆盖不全、CGG重复扩张和CGG重复中的AGG插入无法准确判断、无法确定低比例的嵌合突变会导致临床上存在漏检和误检的问题。通过长片段PCR和高GC PCR相结合同时扩增FXS相关的致病基因FMR1以及FXS中易发生CGG重复扩张和缺失的FMR1的5’-UTR区域及制备三代测序文库，实现全面、精确和快速检测多个样品FXS多种突变的目标。
本发明的第一方面，本发明涉及一种用于FXS多种突变的引物组，包括选自以下的一对或多对引物：
(1)FMR1-E1-F与FMR1-E1-R，其中所述FMR1-E1-F选自SEQ ID NO：1-4，FMR1-E1-R选自SEQ ID NO：5-8；
(2)FMR1-E2-F与FMR1-E9-R，其中所述FMR1-E2-F选自SEQ ID NO：9-11，FMR1-E9-R选自SEQ ID NO：12-14；
(3)FMR1-E10-F与FMR1-E17-R，其中所述FMR1-E10-F选自SEQ ID NO：15-17，FMR1-E17-R选自SEQ ID NO：18-20；
(4)FMR1-Gap-Mix，其中所述FMR1-Gap-Mix选自SEQ ID NO：21-220；
其中，所述FXS多种突变至少包括以下的一种或多种： FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
其中，所述引物位置如图1A至图1B所示。所述引物可以扩增基因组上引物范围内的完整全部序列，包括引物范围内任何类型的突变序列。优选地，每个引物的扩增产物约为2.7kb(FMR1-E1)、11.6kb(FMR1-E2-9)和15.2kb(FMR1-E10-17)。优选地，如果引物位置有SNP，则使用简并碱基引物。
根据一个优选的实施方案，所述引物组包括以下多对引物：
(1)FMR1-E1-F与FMR1-E1-R，其中所述FMR1-E1-F选自SEQ ID NO：1-4，FMR1-E1-R选自SEQ ID NO：5-8；
(2)FMR1-E2-F与FMR1-E9-R，其中所述FMR1-E2-F选自SEQ ID NO：9-11，FMR1-E9-R选自SEQ ID NO：12-14；
(3)FMR1-E10-F与FMR1-E17-R，其中所述FMR1-E10-F选自SEQ ID NO：15-17，FMR1-E17-R选自SEQ ID NO：18-20；和
(4)FMR1-Gap-Mix，其中所述FMR1-Gap-Mix选自SEQ ID NO：21-220。
根据一个优选的实施方案，所述FXS多种突变至少包括以下的一种或多种：FMR1的5’-UTR区域内由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入和微缺失；FMR1的FMR1-Gap-Mix引物范围内的大片段缺失和所有点突变。
根据一个优选的实施方案，本发明的引物组可以同时检测FXS的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、确定5’-UTR区域的微小缺失的准确位点；检测FMR1的全部SNV和FMR1-Gap-Mix引物范围内的大片段缺失。
在一个实施方案中，可以在所述引物的5’端加上5-50nt不同序列的DNA，即DNA条形码(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端的Barcode可以相同或者不同，本领域技术人员可以根据需要选择。
根据一个优选的实施方案，其中所述引物组可以用于1个或2个体系的PCR扩增包括FMR1基因和FMR1基因5’-UTR区域的所有范围内突变类型的FXS相关致病基因片段。再结合后续的PacBio或Nanopore测序平台，可以检测FMR1基因和FMR1基因5’-UTR区域内所有基因片段的突变类型。
本发明第二方面提供了本发明第一方面的引物组在制备用于检测FXS多种突变的试剂盒中的用途，其中所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
本发明第三方面提供了一种用于检测FXS多种突变的试剂盒，包括以下试剂：
(1)用于长片段PCR扩增的试剂；
(2)用于高GC PCR扩增的试剂；
(3)用于构建三代测序文库的试剂。
在一个实施方案中，其中所述用于长片段PCR扩增和高GC PCR扩增的试剂包括DNA聚合酶、反应缓冲液和引物组。
根据一个优选的实施方案，所述试剂盒中的引物组选自以下的一对或多对引物：
(1)FMR1-E1-F与FMR1-E1-R，其中所述FMR1-E1-F选自SEQ ID NO：1-4，FMR1-E1-R选自SEQ ID NO：5-8；
(2)FMR1-E2-F与FMR1-E9-R，其中所述FMR1-E2-F选自SEQ ID NO：9-11，FMR1-E9-R选自SEQ ID NO：12-14；
(3)FMR1-E10-F与FMR1-E17-R，其中所述FMR1-E10-F选自SEQ ID NO：15-17，FMR1-E17-R选自SEQ ID NO：18-20；
(4)FMR1-Gap-Mix，其中所述FMR1-Gap-Mix选自SEQ ID NO：21-220；
其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
其中，所述引物位置如图1A至图1B所示。所述引物可以扩增基因组上引物范围内的完整全部序列，包括引物范围内任何类型的突变序列。优选地，每个引物的扩增产物约为2.7kb(FMR1-E1)、11.6kb(FMR1-E2-9)和15.2kb(FMR1-E10-17)。优选地，如果引物位置有SNP，则使用简并碱基引物。
根据一个优选的实施方案，所述FXS多种突变至少包括以下的一种或多种：FMR1的5’-UTR区域内由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入和微缺失；FMR1的FMR1-Gap-Mix引物范围内的大片段缺失和所有点突变。
根据一个优选的实施方案，本发明的引物组可以同时检测FXS的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、确定5’-UTR区域的微小缺失的准确位点、检测FMR1的全部SNV和FMR1-Gap-Mix引物范围内的大片段缺失。
在一个实施方案中，可以在所述试剂盒中的引物的5’端加入5-50nt不同序列的DNA(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端Barcode可以一样或者不一样，本领域技术人员可以根据需要选择。
在一个实施方案中，对于所述试剂盒，PCR扩增产物在进行下一步反应前，可以纯化或者不纯化，本领域技术人员可以根据需要选择。
在一个实施方案中，其中所述试剂盒中，用于构建三代测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、反应缓冲液和外切酶。
在一个实施方案中，其中对于所述试剂盒，长片段PCR扩增和高GC PCR扩增在一个或两个反应管中完成。
在一个实施方案中，三代测序选自Pacific Biosciences公司的PacBio测序或ONT公司的Nanopore测序。
根据一个优选的实验方案，其中所述用于构建三代PacBio测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇、反应缓冲液和外切酶。
根据一个优选的实施方案，PacBio文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，PacBio通用平末端接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’(SEQ ID NO:221)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio通用TA接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3’(SEQ ID NO:222)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio接头可以带或者不带Barcode。优选地，PacBio接头带PacBio公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述PacBio文库与Pacific Biosciences公司测序平台匹配。
根据一个优选的实施方案，其中所述用于构建三代Nanopore文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇和反应缓冲液。
根据一个优选的实施方案，Nanopore文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，Nanopore接头可以带或者不带Barcode。优选地，Nanopore接头带ONT公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述Nanopore文库与ONT公司测序平台匹配。
本发明第四方面提供了一种用于检测FXS多种突变的系统，包括以下模块：
(1)采集模块：获取受试者样本；
(2)扩增模块：对所述样本进行长片段PCR扩增和高GC PCR扩增；
(3)文库构建模块：构建三代测序文库；
(4)测序模块：测序并分析FMR1基因突变类型。
其中，所述步骤(2)中的PCR扩增采用本发明第一方面所述的引物组进行；
其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
其中，所述引物位置如图1A至图1B所示。所述引物可以扩增基因组上引物范围内的完整全部序列，包括引物范围内任何类型的突变序列。优选地，每个引物的扩增产物约为2.7kb(FMR1-E1)、11.6kb(FMR1-E2-9)和15.2kb(FMR1-E10-17)。优选地，如果引物位置有SNP，则使用简并碱基引物。
在一个实施方案中，其中所述长片段PCR扩增为扩增样本中的FMR1 E2-9、FMR1 E10-17片段。
在一个实施方案中，其中所述高GC PCR扩增为扩增样本中的FMR1 E1片段。
根据一个优选的实施方案，本发明的引物组可以同时检测FXS的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、确定5’-UTR区域的微小缺失的准确位点、检测FMR1的全部SNV和FMR1-Gap-Mix引物范围内的大片段缺失。
在一个实施方案中，可以在所述试剂盒中的引物的5’端加入5-50nt不同序列的DNA(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端Barcode可以一样或者不一样，本领域技术人员可以根据需要选择。
在一个实施方案中，其中所述方法中，长片段PCR和高GC PCR扩增在一个或两个反应管中完成。
在一个实施方案中，其中所述样本选自生物样本或样本提取的gDNA。其中生物样本选自培养的细胞系、血液、羊水、绒毛、配子、囊胚细胞、关节液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液等。
在一个实施方案中，其中所述方法的三代测序选自Pacific Biosciences公司的PacBio测序或ONT公司的Nanopore测序。
根据一个优选的实验方案，其中所述用于构建三代PacBio测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇、反应缓冲液和外切酶。
根据一个优选的实施方案，PacBio文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，PacBio通用平末端接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’(SEQ ID NO:221)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio通用TA接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3’(SEQ ID NO:222)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子，带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio接头可以带或者不带Barcode。在优选的实施方案中，PacBio接头带PacBio公司设计的Barcode或者自行设计的Barcode。本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述PacBio文库与Pacific Biosciences公司测序平台匹配。
根据一个优选的实施方案，其中所述用于构建三代Nanopore文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇和反应缓冲液。
根据一个优选的实施方案，Nanopore文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，Nanopore接头可以带或者不带Barcode，本领域技术人员可以根据需要选择。优选地，Nanopore接头带ONT公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述Nanopore文库与ONT公司测序平台匹配。
本发明第五方面提供了一种用于检测FXS多种突变的方法，包括以下步骤：
(1)获取受试者样本；
(2)对所述样本进行长片段PCR扩增和高GC PCR扩增；
(3)构建三代测序文库；
(4)测序并分析FMR1基因突变类型。
其中，所述步骤(2)中的PCR扩增采用本发明第一方面所述的引物组进行；
其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
其中，所述引物位置如图1A至图1B所示。所述引物可以扩增基因组上引物范围内的完整全部序列，包括引物范围内任何类型的突变序列。优选地，每个引物的扩增产物约为2.7kb(FMR1-E1)、11.6kb(FMR1-E2-9)和15.2kb(FMR1-E10-17)。优选地，如果引物位置有SNP，则使用简并碱基引物。
在一个实施方案中，其中所述长片段PCR扩增为扩增样本中的FMR1 E2-9、FMR1 E10-17片段。
在一个实施方案中，其中所述高GC PCR扩增为扩增样本中的FMR1 E1片段。
根据一个优选的实施方案，本发明的引物组可以同时检测FXS的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、确定5’-UTR区域的微小缺失的准确位点、检测FMR1的全部SNV和FMR1-Gap-Mix引物范围内的大片段缺失。
在一个实施方案中，可以在所述试剂盒中的引物的5’端加入5-50nt不同序列的DNA(Barcode)，用于区分不同的样本；优选地，F和R引物的5’端Barcode可以一样或者不一样，本领域技术人员可以根据需要选择。
在一个实施方案中，其中所述方法中，长片段PCR和高GC PCR扩增在一个或两个反应管中完成。
在一个实施方案中，其中所述样本选自生物样本或样本提取的gDNA。其中生物样本选自培养的细胞系、血液、羊水、绒毛、配子、囊胚细胞、关节液、尿液、汗液、唾液、粪便、脑脊液、腹水、胸水、胆汁或胰腺液等。
在一个实施方案中，其中所述方法的三代测序选自Pacific Biosciences公司的PacBio测序或ONT公司的Nanopore测序。
根据一个优选的实验方案，其中所述用于构建三代PacBio测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇、反应缓冲液和外切酶。
根据一个优选的实施方案，PacBio文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，PacBio通用平末端接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGAT-3’(SEQ ID NO:221)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子。带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio通用TA接头序列为5’-pATCTCTCTCTTTTCCTCCTCCTCCGTTGTTGTTGTTGAGAGAGATT-3’(SEQ ID NO:222)，通过退火形成平末端茎环状结构接头适配子。可以在茎部加上5-50nt不同序列的DNA(Barcode)形成不同带Barcode的接头适配子，带有不同Barcode的PacBio文库可以混合在一起测序。
根据一个优选的实施方案，PacBio接头可以带或者不带Barcode。在优选的实施方案中，PacBio接头带PacBio公司设计的Barcode或者自行设计的Barcode。本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述PacBio文库与Pacific Biosciences公司测序平台匹配。
根据一个优选的实施方案，其中所述用于构建三代Nanopore文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、80％乙醇和反应缓冲液。
根据一个优选的实施方案，Nanopore文库接头连接可以使用平末端连接或TA连接的方式。
根据一个优选的实施方案，Nanopore接头可以带或者不带Barcode，本领域技术人员可以根据需要选择。优选地，Nanopore接头带ONT公司设计的Barcode或者自行设计的Barcode，本领域技术人员可以根据需要选择。
根据一个优选的实施方案，所述Nanopore文库与ONT公司测序平台匹配。
基于长片段PCR和高GC PCR扩增和第三代高通量测序特定组合的本发明所述的方法可高特异性地、准确和快速地实现同时检测多个样品FXS相关致病基因的多种突变。
本发明所述方法和试剂盒的优异技术效果主要在于以下几个方面：
(1)检测范围广：本发明可以同时检测目前已研究发现的FXS的CGG重复扩张类型，以及可以检测发生在CGG重复区域的缺失；可以检测FMR1基因的全部SNV和FMR1基因上下游约2MB区域内的大片段缺失。
(2)多种突变类型单个试剂盒检测：传统的方法针对每种突变类型都需要设置一种检测体系，而本发明在两个反应引物体系里同时检测多种突变，包括由CGG扩张引起的前突变和全突变、判断CGG重复中的AGG插入、微缺失、SNV和大片段缺失。
(3)检测误检和漏检率低：目前通用的检测FXS的CGG扩张最常见的方法是结合CE的PCR反应检测，对于一些低比例的嵌合突变由于信号噪音的影响容易判断为阴性，或者将阴性判断为突变患者，而本发明的高GC PCR体系结合三代测序的方式可以排除噪音的影响，更准确的确认FXS中的CGG扩张类型；而目前并没有对FMR1基因5’-UTR区域的微缺失和FMR1基因的SNV检测形成有效的方案，因此，一些由于这些原因造成的突变无法被检测发现，本发明的两个反应引物体系可以同时检测上述突变类型。
(4)样本多样化：用于PCR的模板可以是外周血、干血斑或经提取的基因组DNA，也可以是人源细胞系或其他特定的组织。
(5)高通量检测：三代测序可实现384种Barcode接头，实际还可以根据需要设计更多种Barcode接头。或者利用引物带Barcode和接头带Barcode的双Barcode系统实现更多种Barcode组合。三代测序平台的高通量特性决定可以实现高通量样品检测。
(6)精确度高：PacBio的哑铃状文库在测序时可进行多轮解读，矫正后测序结果碱基精确度大于99％。而且PacBio测序错误是随机的，再通过测序深度矫正碱基精确度大于99.9％。因此可以精确解读引物检测范围内的基因突变。
(7)检测时间灵活：Nanopore平台可在数分钟内产生数据，可根据实际数据量需求在数分钟或数小时内开启数据分析。当对检测时效要求比较高时，Nanopore平台具有时间优势。
附图说明
图1A-1B：PCR引物设计示意图，其中图1A表示FXS相关FMR1 E1检测引物设计示意图，和图1B表示FXS相关FMR1基因和大片段缺失检测引物设计示意图。
图2A-2B：FXS扩增片段凝胶电泳示意图，其中图2A表示长片段PCR扩增FMR1 E2-9和FMR1 E10-17的DNA凝胶电泳示意图，和图2B表示高GC PCR扩增FMR1 E1的DNA凝胶电泳示意图。
图3A-3L：代表性的FXS相关基因突变样本PacBio测序结果图，其中图3A表示正常人样本CGG扩张(29)，图3B表示前突变携带者样本CGG扩张(29/87)，图3C表示全突变携带者样本CGG扩张(36/439)，图3D表示正常人样本CGG扩张(30)，图3E表示前突变携带者样本CGG扩张(29/118)，图3F表示全突变样本CGG扩张(203/342/725)，图3G表示前突变携带者样本CGG扩张(29/61)，图3H表示全突变样本CGG扩张(335/659)，图3I表示前突和全突变嵌合样本CGG扩张(69/191/221/336)，图3J表示FMR1点突变样本，图3K表示FMR1 5’-UTR微缺失样本，和图3L表示FMR1大片段缺失样本。
具体实施方式
实施例1：利用本发明涉及的PCR方法扩增并构建PacBio测序文库
步骤1：长片段和高GC PCR扩增
按照下表1(长片段PCR)和表2(高GC PCR)制备反应体系，扩增外周血、干血斑和基因组DNA样本：
表1：

表2：
在PCR仪上，按照下表3所示条件进行预扩增：
表3：

扩增完成后，每个样本取5ul，在1％的DNA凝胶上检测，结果如图2所示，以不同样本为模板，FMR1基因不同片段均能得到有效地扩增；同时，将扩增产物放入离心机中，10000rpm，离心20min。离心结束后水平静置放置，取4μL上清加入新的管内，进行下一步实验。
步骤2：构建PacBio测序文库
按照下表4制备反应体系：
表4：
在PCR仪上，按如下条件进行反应：37℃20min；25℃15min；65℃10min。反应完成后，加入0.5μL Exonuclease  III(NEB,Cat#M0206L)和0.5μL Exonuclease VII(NEB,Cat#M0379L)，继续在37℃反应1小时。用0.6x Ampure PB磁珠(PacBio,Cat#100-265-900)依照制造商的说明书纯化两次，最后用10uL Elution Buffer洗脱DNA。所得DNA洗脱液即是目标DNAPacBio测序文库。用Qubit dsDNA HS试剂(ThermoFisher,Cat#Q32851)在Qubit 3 Fluoromter(ThermoFisher,Cat#Q33216)上测定DNA浓度。当有多个样本PacBio测序文库时，可以取等量的文库混合在一起，制备成混合文库。
步骤3：PacBio上机测序和分析
根据文库的总浓度与摩尔浓度，将适当体积的文库与结合试剂(PacBio，Cat#101-820-200)和引物(PacBio,Cat#100-970-100)反应，制备成最终可上机文库。代表性测序结果如图3所示，其中图3A表示正常人样本(CGG重复数为29)，图3B表示前突变携带者样本(CGG重复数为29和87),图3C表示全突变携带者样本CGG扩张(36/439)。
实施例2：利用本发明涉及的PCR方法构建PacBio测序文库
步骤1：长片段和高GC PCR扩增
按照下表5(长片段PCR)和表6(高GC PCR)制备反应体系，扩增不同类型FXS相关基因突变的外周血样本：
表5：

表6：
在PCR仪上，按照下表7所示条件进行预扩增：
表7：
扩增完成后，将扩增产物放入离心机中，10000rpm，离心20min。离心结束后水平静置放置，取4μL上清加入新的管内。
步骤2：构建PacBio测序文库
按照下表8制备反应体系：
表8：
在PCR仪上，按如下条件进行反应：37℃20min；25℃15min；65℃10min。反应完成后，加入0.5μL Exonuclease III(NEB,Cat#M0206L)和0.5μL Exonuclease VII(NEB,Cat#M0379L)，继续在37℃反应1小时。用0.6x Ampure PB磁珠(PacBio,Cat#100-265-900)依照制造商的说明书纯化两次，最后用10uL Elution Buffer洗脱DNA。所得DNA洗脱液即是目标DNAPacBio测序文库。用Qubit dsDNA HS试剂(ThermoFisher,Cat#Q32851)在Qubit 3 Fluoromter(ThermoFisher,Cat#Q33216)上测定DNA浓度。当有多个样本PacBio测序文库时，可以取等量的文库混合在一起，制备成混合文库。
步骤3：PacBio上机测序和分析
根据文库的总浓度与摩尔浓度，将适当体积的文库与结合试剂(PacBio，Cat#101-820-200)和引物(PacBio,Cat#100-970-100)反应，制备成最终可上机文库。代表性测序结果如图3所示，图3D表示正常人样本CGG扩张(30)，图3E表示前突变携带者样本CGG扩张(29/118)，图3F表示全突变样本CGG扩张(203/342/725)。
实施例3：利用本发明涉及的PCR方法构建PacBio测序文库
步骤1：长片段和高GC PCR扩增
按照下表9(长片段PCR)和表10(高GC PCR)制备反应体系，扩增不同类型FXS相关基因突变的外周血样本：
表9：

表10：
在PCR仪上，按照下表11所示条件进行预扩增：
表11：
扩增完成后，将扩增产物放入离心机中，10000rpm，离心20min。离心结束后水平静置放置，取4μL上清加入新的管内。
步骤2：构建PacBio测序文库
按照下表12制备反应体系：
表12：
在PCR仪上，按如下条件进行反应：37℃20min；25℃15min；65℃10min。反应完成后，加入0.5μL Exonuclease III(NEB,Cat#M0206L)和0.5μL Exonuclease VII(NEB,Cat#M0379L)，继续在37℃反应1小时。用0.6x Ampure PB磁珠(PacBio,Cat#100-265-900)依照制造商的说明书纯化两次，最后用10uL Elution Buffer洗脱DNA。所得DNA洗脱液即是目标DNAPacBio测序文库。用Qubit dsDNA HS试剂(ThermoFisher,Cat#Q32851)在Qubit 3 Fluoromter(ThermoFisher,Cat#Q33216)上测定DNA浓度。当有多个样本PacBio测序文库时，可以取等量的文库混合在一起，制备成混合文库。
步骤3：PacBio上机测序和分析
根据文库的总浓度与摩尔浓度，将适当体积的文库与结合试剂(PacBio，Cat#101-820-200)和引物(PacBio,Cat#100-970-100)反应，制备成最终可上机文库。代表性测序结果如图3所示，图3G表示前突变携带者样本CGG扩张(29/61)，图3H表示全突变样本CGG扩张(335/659)，图3I表示前突和全突变嵌合样本CGG扩张(69/191/221/336)，图3J表示FMR1点突变样本，图3K表示FMR1 5’-UTR微缺失样本，图3L表示FMR1大片段缺失样本。
实施例4：FXS基因突变的检测和验证
收集24个受试者的外周血基因组DNA作为验证样品24例，参照实施例3，利用本发明方法(和试剂盒)同时检测FXS相关基因的多种突变。同时用PCR-CE的方法检测FXS的CGG重复扩张，结合Sanger测序的方法检测相关基因的点突变和缺失。利用本发明得到的结果和对照结果相对比，结果如表13所示，24例样本结果完全一致。
表13

因此，利用本发明方法检测的结果，经过与PCR-CE结合PCR-Sanger测序方法对比，特异性和灵敏度均达到100％。本发明方法可以直接检测CGG重复序列，而不是通过其他方式进行推测；因此，本发明方法的准确性优于PCR-CE法；而且24例样本中，有7例样本通过本发明的方法确定全突变样本的CGG重复数。本发明方法可以直接检测未知的FMR1的全部SNV和大片段缺失，而PCR-sanger法只能对已知结果进行验证。
应当理解，尽管本发明已根据其优选实施例进行了示例性描述，但不应限于上述实施例，对于本领域的技术人员来说，本发明可以有各种更改和变化。长片段PCR和高GC PCR反应和三代测序文库构建中所涉及的反应试剂、反应条件等等可以根据具体的需要进行相应的调整和改变。因此对于本领域技术人员来说，在不脱离本发明的构思和原则之内，还可做出若干简单替换，这些均应包含在本发明的保护范围之内。
参考文献
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[6]Collins S C,Bray S M,Suhl J A,et al.Identification of novel FMR1 variants by massively parallel sequencing in developmentally delayed males.American Journal of Medical Genetics Part A,2010,152(10):2512-2520.doi:10.1002/ajmg.a.33626.
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Claims (19)

  1. 一种用于扩增FXS多种突变的引物组，包括选自以下的一对或多对引物：
    (1)FMR1-E1-F与FMR1-E1-R，其中所述FMR1-E1-F选自SEQ ID NO：1-4，FMR1-E1-R选自SEQ ID NO：5-8；
    (2)FMR1-E2-F与FMR1-E9-R，其中所述FMR1-E2-F选自SEQ ID NO：9-11，FMR1-E9-R选自SEQ ID NO：12-14；
    (3)FMR1-E10-F与FMR1-E17-R，其中所述FMR1-E10-F选自SEQ ID NO：15-17，FMR1-E17-R选自SEQ ID NO：18-20；和
    (4)FMR1-Gap-Mix，其中所述FMR1-Gap-Mix选自SEQ ID NO：21-220；
    其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
  2. 根据权利要求1所述的引物组，其中，所述引物在5’端包括5-50nt不同序列的DNA条形码。
  3. 权利要求1-2任一项所述的引物组在制备用于检测FXS多种突变种的试剂盒中的用途，其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
  4. 一种用于检测FXS多种突变的试剂盒，包括以下试剂：
    (1)用于长片段PCR扩增的试剂；
    (2)用于高GC PCR扩增的试剂；
    (3)用于构建三代测序文库的试剂；
    其中，所述用于PCR扩增的试剂包含权利要求1-2任一项所述的引物组；
    其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
  5. 根据权利要求4所述的试剂盒，其中，所述长片段PCR扩增和高GC PCR扩增在一个或者两个反应管中完成。
  6. 根据权利要求4所述的试剂盒，其中，所述用于长片段PCR扩增的试剂包括DNA聚合酶、反应缓冲液和引物。
  7. 根据权利要求4所述的试剂盒，其中，所述用于高GC PCR扩增的试剂包括DNA聚合酶、反应缓冲液和引物。
  8. 根据权利要求4所述的试剂盒，其中，所述用于构建三代测序文库的试剂包括末端修复酶、接头、连接酶、DNA纯化磁珠、反应缓冲液和外切酶。
  9. 根据权利要求4所述的试剂盒，其中，所述三代测序选自Pacific Biosciences公司的PacBio测序或Oxford Nanopore Technologies公司的Nanopore测序。
  10. 一种用于检测FXS多种突变的系统，包括以下模块：
    (1)采集模块：获取受试者样本；
    (2)扩增模块：对所述样本进行长片段PCR扩增和高GC PCR扩增；
    (3)文库构建模块：构建三代测序文库；
    (4)测序模块：测序并分析FMR1基因突变类型；
    其中，所述模块(2)中的PCR扩增采用权利要求1-2任一项所述的引物组进行；
    其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
  11. 根据权利要求10所述的系统，其中，所述长片段PCR扩增样本中的FMR1E2-9、FMR1E10-17片段。
  12. 根据权利要求10所述的系统，其中，所述高GC PCR扩增样本中的FMR1E1片段。
  13. 根据权利要求10所述的系统，其中，所述长片段PCR扩增和高GC PCR扩增在一个或者两个反应管中完成。
  14. 根据权利要求10所述的系统，其中，所述三代测序选自Pacific Biosciences公司的PacBio测序或Oxford Nanopore Technologies公司的Nanopore测序。
  15. 一种用于检测FXS多种突变的方法，包括以下步骤：
    (1)获取受试者样本；
    (2)对所述样本进行长片段PCR扩增和高GC PCR扩增；
    (3)构建三代测序文库；
    (4)测序并分析FMR1基因突变类型；
    其中，所述步骤(2)中的PCR扩增采用权利要求1-2任一项所述的引物组进行；
    其中，所述FXS多种突变至少包括以下的一种或多种：FMR1的由CGG扩张引起的前突变和全突变、CGG重复中的AGG插入、大片段缺失、点突变和微缺失。
  16. 根据权利要求15所述的方法，其中，所述长片段PCR扩增样本中的FMR1E2-9、FMR1E10-17片段。
  17. 根据权利要求15所述的方法，其中，所述高GC PCR扩增样本中的FMR1E1片段。
  18. 根据权利要求15所述的方法，其中，所述长片段PCR扩增和高GC PCR扩增在一个或者两个反应管中完成。
  19. 根据权利要求15所述的方法，其中，所述三代测序选自Pacific Biosciences公司的PacBio测序或Oxford Nanopore Technologies公司的Nanopore测序。
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