CN111500679A - Preparation method of long fragment capture sequencing probe set - Google Patents

Preparation method of long fragment capture sequencing probe set Download PDF

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CN111500679A
CN111500679A CN202010589532.9A CN202010589532A CN111500679A CN 111500679 A CN111500679 A CN 111500679A CN 202010589532 A CN202010589532 A CN 202010589532A CN 111500679 A CN111500679 A CN 111500679A
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chrx
gene
probe
chr9
chr16
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潘世让
王洋
梁羽
吴昕
汪德鹏
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Grandomics Biosciences Co ltd
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    • 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
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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
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation
    • 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
    • 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

Abstract

The invention belongs to the technical field of biology, and particularly relates to a preparation method of a long fragment capture sequencing probe set.

Description

Preparation method of long fragment capture sequencing probe set
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a preparation method of a long fragment capture sequencing probe set.
Background
The human genome is approximately 3G in size and contains about 2 ten thousand protein-encoding genes. The whole genome sequencing needs to consume a great deal of cost and time, and the target sequence capture sequencing becomes a hot technology in the current genomics research. When sequencing only the target sequence, researchers can measure a greater number of samples and a greater depth at the same cost. Especially for some rare variants or gene mutation of partial somatic cells, the sequencing depth determines that the target sequence capture sequencing is an effective tool.
The target sequence capture sequencing is to customize a target genome region of interest into a specific probe to hybridize with genome DNA, enrich DNA fragments of the target genome region and then sequence.
The target enrichment based on the capture of the liquid phase hybridization probe is to capture a target region by thousands to millions of oligonucleotide capture probes which are designed to be complementary with the target region and then sequence the target region, and the method is characterized in that the design of the probe is flexible, the flux is high, the capture region can be large or small, and the fusion gene can be detected; the disadvantage is the relatively high cost of probe synthesis, especially for large panels. Therefore, if the number of probes in Panel can be reduced, the popularization and application of the long fragment sequence determination method can be effectively promoted.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide an economical and effective long fragment capture sequencing method capable of reducing the number of probes.
The invention relates to a preparation method of a long fragment capture sequencing probe set, which comprises the following steps:
a) the average probe spacing range was calculated using the following formula:
N＝(L+2)×P±3×P；
wherein:
n: average probe spacing in bp;
p: average length of each probe, unit bp;
l genome fragmentation average length unit Kb, taking an integer;
b) preparing a probe set for detecting the target region according to the average interval range of the probes.
The method for designing the probe has the advantages of comprehensiveness, rapidness, accuracy and high cost performance, and solves the problems of intensive capture sequencing probes and high synthesis cost.
According to another aspect of the invention, the invention also relates to the use of the method as described above in third generation sequencing.
According to another aspect of the invention, the invention also relates to the probe set prepared by the method as described above, or a kit comprising the probe set, and the use of the probe set or the kit.
The probe set prepared by the method provided by the invention can detect various mutation information of a target gene, and is simultaneously suitable for detecting various tissue samples such as muscle, blood, amniotic fluid and the like.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is an average depth of coverage of 0.5 × for a captured DNA fragment having an average length of 5Kb for genomic fragmentation in one embodiment of the present invention;
FIG. 2 is an average depth of coverage of 0.5 × for a captured DNA fragment with an average length of 2Kb for genomic fragmentation in one embodiment of the present invention;
FIG. 3 is an average depth of coverage of 0.5 × for a captured DNA fragment having an average length of 8Kb for genomic fragmentation in one embodiment of the present invention;
FIG. 4 is a graph of probe addition versus target rate in capture for one embodiment of the present invention.
Detailed Description
The invention relates to a preparation method of a long fragment capture sequencing probe set, which comprises the following steps:
a) the average probe spacing range was calculated using the following formula:
N＝(L+2)×P±3×P；
wherein:
n: average probe spacing in bp;
p: average length of each probe, unit bp;
l genome fragmentation average length unit Kb, taking an integer;
b) preparing a probe set for detecting the target region according to the average interval range of the probes.
A "target region" of a genome (and any grammatical equivalents thereof) refers to any one or more regions of the genome as a whole or identified as a target and/or selected genome by one or more of the methods described herein. Target regions of a genome sequenced by the methods and systems described herein include, but are not limited to, introns, exons, intergenic regions, or any combination thereof. In certain examples, the methods and systems described herein provide sequence information about a full exome, a portion of an exon, one or more selected genes (including a selected set of genes), one or more introns, and a combination of intronic and exonic sequences.
The target region of the genome may also include certain portions or percentages of the genome rather than regions identified by sequence. In certain embodiments, the target region of the genome captured and analyzed according to the methods described herein comprises a portion of the genome located at every 1, 2, 5, 10, 15, 20, 25, 50, 100, 200, 250, 500, 750, 1000, or 10000Kb of the genome. In other embodiments, the target region of the genome comprises 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of the whole genome. In other embodiments, the target region comprises 1-10%, 5-20%, 10-30%, 15-40%, 20-50%, 25-60%, 30-70%, 35-80%, 40-90%, or 45-95% of the whole genome. Furthermore, the target region may be continuous or intermittent.
In some embodiments, the value of P ranges from 15 to 250 bp; such as 15bp, 20bp, 30bp, 40bp, 50bp, 60bp, 70bp, 80bp, 90bp, 100bp, 110bp, 120bp, 130bp, 140bp, 150bp, 160bp, 170bp, 180bp, 190bp, 200bp, 210bp, 220bp, 230bp, 240bp, 250bp, or a range value composed of the above values.
In some embodiments, the probe is used to detect a target region of no less than 1Kb in length, optionally 1.5Kb, 2Kb, 2.5Kb, 3Kb, 4Kb, 5Kb, 10Kb, 15Kb, 20Kb, 30Kb, 40Kb, 50Kb, 100Kb, 150Kb, 200Kb, 300Kb, 400Kb, 500Kb, 600Kb, 700Kb, 800Kb, 900Kb, 1mb, 2mb, 3mb, 4mb, 5mb, 10mb, 15mb, 20mb or more.
Probes in a probe set should be complementary paired to corresponding regions in the target region. The term "complementary pair" as used herein means that the probe is capable of hybridizing to the desired corresponding site under stringent conditions. The stringent conditions used in the present invention are well known and include, for example, hybridization at 65 ℃ for 12 to 16 hours in a hybridization solution containing 400mM NaCl, 40mM PIPES (pH6.4) and 1mM EDTA, followed by washing at 65 ℃ for 15 to 60 minutes with a washing solution containing 0.1% SDS and 0.1% SSC. This is familiar to the person skilled in the art.
In some embodiments, the value of L is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
In some embodiments, the species of the target region may be selected from animals including humans and all animal breeds (e.g., livestock and pets) and wild animals and birds including, without limitation, cows, horses, cows, pigs, sheep, goats, rats, mice, dogs, cats, rabbits, camels, donkeys, deer, mink, chickens, ducks, geese, turkeys, etc., with mammals being preferred, primates being preferred, and humans being most preferred.
In some embodiments, the method further comprises grouping the sets of probes, the grouping selected from at least one of the following groups:
(1) capturing the probe for the full length of the target region;
(2) a probe that captures only an exon region of the target region;
(3) probes that capture only the high mutation rate region.
In some embodiments, the method further comprises coupling the probe to a label bound to a solid phase, the label being selected from one or more of streptavidin, biotin, avidin, an antibody, a chemical coupling agent, preferably biotin.
In the present invention, the term "target region" or "gene" is understood to include, but not limited to, any of the (preferably human) Dystrophin gene, TSC1 gene, TSC2 gene, BRAC1 gene, BRAC2 gene, GJB2 gene, 12SrRNA gene, or S L C26A4 gene.
The invention also relates to the application of the method in the third generation sequencing.
In some embodiments, the third generation sequencing is performed using a PacBio sequence, PromethION, MinION, or gridios platform.
The invention also relates to a probe set, which is prepared by the method;
in some embodiments, the probe set is used to detect the TSC (tuberous sclerosisomicplex) gene, which is used to refer to the TSC1 gene and/or the TSC2 gene herein unless otherwise specified.
In some specific embodiments, the probe set comprises probes that are complementary paired to the following chromosomal regions (hereinafter referred to as TSC probe sets):
chr9:135813137-135813237,chr9:135789857-135789957,chr9:135768617-135768717,chr9:135768977-135769077,chr9:135769457-135769557,chr9:135769817-135769917,chr9:135770297-135770397,chr9:135770777-135770877,chr9:135771137-135771237,chr9:135771617-135771717,chr9:135772097-135772197,chr9:135772457-135772557,chr9:135772937-135773037,chr9:135758177-135758277,chr9:135773777-135773877,chr9:135774257-135774357,chr9:135774617-135774717,chr9:135775097-135775197,chr9:135775577-135775677,chr9:135775937-135776037,chr9:135776417-135776517,chr9:135776897-135776997,chr9:135777257-135777357,chr9:135777737-135777837,chr9:135758657-135758757,chr9:135778577-135778677,chr9:135779057-135779157,chr9:135779417-135779517,chr9:135779897-135779997,chr9:135780377-135780477,chr9:135780617-135780717,chr9:135781097-135781197,chr9:135781577-135781677,chr9:135781937-135782037,chr9:135782417-135782517,chr9:135783737-135783837,chr9:135759257-135759357,chr9:135784577-135784677,chr9:135785057-135785157,chr9:135785897-135785997,chr9:135786377-135786477,chr9:135786737-135786837,chr9:135787217-135787317,chr9:135787697-135787797,chr9:135788057-135788157,chr9:135759737-135759837,chr9:135790217-135790317,chr9:135790697-135790797,chr9:135791177-135791277,chr9:135791537-135791637,chr9:135792017-135792117,chr9:135792497-135792597,chr9:135793217-135793317,chr9:135793697-135793797,chr9:135794057-135794157,chr9:135794537-135794637,chr9:135795377-135795477,chr9:135795857-135795957,chr9:135796217-135796317,chr9:135796697-135796797,chr9:135797177-135797277,chr9:135797537-135797637,chr9:135798017-135798117,chr9:135798497-135798597,chr9:135798857-135798957,chr9:135799337-135799437,chr9:135800177-135800277,chr9:135800657-135800757,chr9:135801017-135801117,chr9:135801497-135801597,chr9:135801977-135802077,chr9:135802337-135802437,chr9:135802817-135802917,chr9:135803297-135803397,chr9:135803657-135803757,chr9:135804137-135804237,chr9:135804857-135804957,chr9:135805337-135805437,chr9:135761417-135761517,chr9:135806177-135806277,chr9:135806657-135806757,chr9:135807017-135807117,chr9:135807497-135807597,chr9:135807977-135808077,chr9:135808337-135808437,chr9:135808817-135808917,chr9:135809297-135809397,chr9:135809657-135809757,chr9:135810137-135810237,chr9:135761897-135761997,chr9:135810977-135811077,chr9:135811457-135811557,chr9:135811817-135811917,chr9:135812297-135812397,chr9:135813617-135813717,chr9:135814097-135814197,chr9:135814457-135814557,chr9:135814937-135815037,chr9:135815777-135815877,chr9:135816257-135816357,chr9:135816977-135817077,chr9:135817457-135817557,chr9:135817817-135817917,chr9:135818297-135818397,chr9:135818777-135818877,chr9:135819137-135819237,chr9:135819617-135819717,chr9:135820097-135820197,chr9:135820457-135820557,chr9:135820937-135821037,chr9:135762977-135763077,chr9:135821777-135821877,chr9:135822257-135822357,chr9:135822617-135822717,chr9:135823577-135823677,chr9:135823937-135824037,chr9:135824417-135824517,chr9:135824897-135824997,chr9:135825737-135825837,chr9:135763457-135763557,chr9:135826577-135826677,chr9:135827057-135827157,chr9:135828377-135828477,chr9:135828617-135828717,chr9:135765137-135765237,chr9:135765617-135765717,chr9:135766097-135766197,chr9:135766457-135766557,chr9:135766937-135767037,chr9:135757577-135757677,chr9:135767777-135767877,chr9:135768257-135768357,chr16:2144932-2145032,chr16:2088172-2088272,chr16:2117692-2117792,chr16:2102452-2102552,chr16:2107732-2107832,chr16:2147572-2147672,chr16:2147812-2147912,chr16:2091412-2091512,chr16:2116732-2116832,chr16:2099812-2099912,chr16:2100292-2100392,chr16:2101612-2101712,chr16:2102092-2102192,chr16:2102932-2103032,chr16:2089252-2089352,chr16:2103772-2103872,chr16:2104252-2104352,chr16:2104612-2104712,chr16:2105092-2105192,chr16:2105932-2106032,chr16:2106412-2106512,chr16:2106892-2106992,chr16:2107252-2107352,chr16:2089732-2089832,chr16:2108572-2108672,chr16:2110372-2110472,chr16:2110732-2110832,chr16:2111212-2111312,chr16:2111692-2111792,chr16:2111932-2112032,chr16:2112412-2112512,chr16:2112892-2112992,chr16:2113252-2113352,chr16:2113732-2113832,chr16:2090332-2090432,chr16:2114572-2114672,chr16:2115412-2115512,chr16:2115892-2115992,chr16:2116372-2116472,chr16:2117212-2117312,chr16:2118532-2118632,chr16:2090812-2090912,chr16:2120212-2120312,chr16:2120692-2120792,chr16:2121172-2121272,chr16:2121532-2121632,chr16:2122012-2122112,chr16:2122492-2122592,chr16:2122852-2122952,chr16:2123332-2123432,chr16:2091292-2091392,chr16:2124052-2124152,chr16:2124532-2124632,chr16:2125852-2125952,chr16:2126212-2126312,chr16:2126692-2126792,chr16:2127172-2127272,chr16:2127532-2127632,chr16:2128012-2128112,chr16:2128492-2128592,chr16:2128852-2128952,chr16:2129332-2129432,chr16:2130172-2130272,chr16:2131012-2131112,chr16:2131492-2131592,chr16:2131972-2132072,chr16:2132332-2132432,chr16:2132812-2132912,chr16:2133292-2133392,chr16:2133652-2133752,chr16:2134132-2134232,chr16:2092372-2092472,chr16:2134972-2135072,chr16:2135452-2135552,chr16:2136172-2136272,chr16:2136652-2136752,chr16:2137012-2137112,chr16:2137492-2137592,chr16:2137972-2138072,chr16:2138332-2138432,chr16:2138812-2138912,chr16:2139292-2139392,chr16:2139652-2139752,chr16:2140132-2140232,chr16:2092972-2093072,chr16:2140972-2141072,chr16:2141452-2141552,chr16:2141812-2141912,chr16:2142292-2142392,chr16:2142772-2142872,chr16:2143132-2143232,chr16:2143612-2143712,chr16:2144092-2144192,chr16:2144452-2144552,chr16:2093452-2093552,chr16:2145772-2145872,chr16:2146252-2146352,chr16:2147092-2147192,chr16:2093812-2093912,chr16:2094292-2094392,chr16:2094772-2094872,chr16:2096092-2096192,chr16:2096452-2096552,chr16:2096932-2097032,chr16:2088652-2088752,chr16:2097772-2097872,chr16:2098252-2098352,chr16:2098612-2098712
wherein the chromosome region is a region on the genes of the human TSC1 and TSC2, and the reference sequence is the human reference genome version Dec.2013(GRCh38/hg38) (TSC1: chr9:132891348-132944633 and TSC2: chr16: 2047895-2088720).
The invention also relates to a kit for gene detection, comprising a probe set as described above.
In some embodiments, the kit further comprises one or more of a solid support as defined above, a linker sequence, primers for binding to the linker sequence and amplifying a nucleic acid fragment, a DNA extraction system, a PCR reaction buffer, nuclease-free water, a DNA polymerase, a molecular weight marker, a target sequence eluent, a terminal repair enzyme, a terminal repair buffer, a DNA ligase.
In some embodiments, the probes in the set of probes further comprise a label that is bindable to a solid phase; in some embodiments, the label may be selected from the group consisting of biotin, streptavidin, avidin, an antibody, a chemical coupling agent; and any biological, chemical, physical or enzymatic reagents known in the art for affinity purification.
"chemical coupling agent" refers to a group that is capable of reacting with another chemical group to form a covalent bond, i.e., a group that is covalently reactive under suitable reaction conditions. Generally, nucleophilic groups, electrophilic groups, and photoactivatable groups can be selected. Exemplary chemical coupling agents include, but are not limited to, olefins, acetylenes, alcohols, acids, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines, hydrazines, hydrazones, hydrazides, diazos, diazonium salts, niter (nitre), nitriles, thiols, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids, sulfuric acids, acetals, ketals, anhydrides, sulfates, sulfenamides, amidines, diimides, imides, nitrones, hydroxylamines, oximes, hydroxamic acids, thiohydroxamic acids, allenes, orthoesters, sulfites, enamines, acetylenic amines, ureas, pseudoureas, semicarbazides, carbodiimides, carbamates, imines, azides, azo compounds, azoxy compounds, and nitroso compounds. Reactive functional groups of chemical coupling agents also include those used to prepare bioconjugates, such as N-hydroxysuccinimide esters, maleimides, and the like. Methods of preparing each of these Functional groups are well known in the art and their use or modification for a particular purpose is within the ability of those skilled in the art (see, e.g., Sandier and Karo, editors, Organic Functional group precursors. academic Press, San Diego, 1989).
The probe can be combined with third-generation sequencing to achieve better technical effects.
The invention also relates to a gene sequencing method, which comprises the following steps:
A) breaking the genome DNA in a sample to be detected into nucleic acid fragments and amplifying to construct a DNA library;
B) capturing in the DNA library a target sequence capable of specifically binding to the probe set using the probe set as described above;
C) sequencing the target sequence.
In some embodiments, when N is 7P, the amount of probe added to the probe set is 0.3pmol or more, for example 0.3 to 0.8 pmol.
In some embodiments, the sample to be tested is a bodily fluid, tissue, or tissue lysate; the body fluid may be selected from, for example, blood (whole blood), serum, plasma, cell culture supernatant, saliva, semen, tissue or tissue lysate; the tissue may be selected from, for example, amniotic fluid, villi, bone, muscle, or hair, among others.
In some embodiments, the method of constructing a DNA library comprises:
and (3) connecting the nucleic acid fragment with an adaptor sequence after the end of the nucleic acid fragment is repaired, and designing a primer by taking the adaptor sequence as a template to carry out PCR amplification.
In some embodiments, the capturing occurs on a solid support;
the solid phase carrier is preferably enrichment particles, and the enrichment particles are coated with biotin or avidin; preferably avidin, and the object captured by it is biotin.
In a preferred embodiment, the "solid support" is an "enrichment particle"; as used herein, "enriched particles" refers to discrete small objects, such as spheres (e.g., beads), capsules, polyhedrons, and the like, that can be of various shapes. The particles may be macroscopic or microscopic, such as microparticles or nanoparticles. The particles may be non-magnetic or magnetic. The magnetic particles may contain a ferromagnetic substance, and the ferromagnetic substance may be Fe, Ni, Co, iron oxide, or the like.
In some embodiments, the target sequence is amplified prior to sequencing the target sequence.
In some embodiments, the amplification is specifically a PCR amplification.
In some embodiments, the sequencing is third generation sequencing;
in some embodiments, the third generation sequencing is performed using a PacBio sequence, PromethION, MinION, gridios platform.
More preferred embodiments are magnetic beads.
According to one aspect of the invention, the invention also relates to the use of a probe or probe combination as described above, or a kit as described above, or a method as described above, for detecting a variation in a human gene (e.g. a TSC gene);
in some embodiments, the human genetic (e.g., TSC gene) variation comprises an insertion, deletion, replication, inversion, translocation, SNP.
This application includes diagnostic and non-diagnostic purposes, such as in genetic studies, race distribution, human chemistry, etc. (typically the application of SNPs), or may be the identification of cellular and animal models of TSC gene-related diseases if homology is high for humans.
According to one aspect of the invention, the invention also relates to the use of a panel of probes as described above, or a kit as described above, for the preparation of a diagnostic agent for tuberous sclerosis in humans.
Embodiments of the present invention will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
Example 1
The Dystrophin gene (DMD gene for short) is one of the largest human genes, and is located at Xp21 and has a length of about 2.3 Mb. The coding region of the gene comprises 79 exons and 7 tissue-specific promoters, and the mRNA sequence of the gene is 14Kb in length. The most common mutation of the Dystrophin gene is a deletion of one or more exons within the gene, accounting for 65% of the total mutation pattern. The incidence rate of repeated mutation is 5-15% due to the different sensitivity of detection means. The remaining mutations are point mutations or microdeletions, which usually result in nonsense and frameshift mutations. Among mutations in the dysophilin gene, about 30% are new mutations, not genetic ones. Among them, mutations that cause dystrophin deficiency encoded by this gene cause the common neuromuscular disease dystrophin disease (dystrophanopathy). The disease is classified into Duchenne Muscular Dystrophy (DMD) and Becker Muscular Dystrophy (BMD) according to the age of onset, the presence or absence of pseudohypertrophy of muscle, the course of disease, the prognosis, and the like.
The method of the invention is used for determining the average interval range of the probes for the DMD gene, the average length L of genome fragmentation is set to be 5Kb, the average length P of each probe is set to be 120bp, the average interval N of the probes is calculated to be 480-1200 bp. by substituting the formula N (L +2) × P +/-3 × P, 1000bp is selected as the average interval of the probes in the range, when the DMD gene is designed, the GC content value of the probes and the hairclip-free structure are ensured at the same time, Blast comparison is further carried out with databases such as NCBI, and the probes are ensured to avoid the repeated region on the genome so as to ensure the specificity of the probes.
The Dystrophin probe groupings were as follows:
group I:
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8,chrX:33357475-33357595,chrX:33358559-33358679,chrX:33359427-33359547,chrX:33360506-33360626,chrX:33361581-33361701,chrX:33362604-33362724,chrX:33363429-33363549,chrX:33365538-33365658,chrX:33366519-33366639,chrX:33367361-33367481,chrX:33368498-33368618,chrX:33369408-33369528,chrX:33370588-33370708,chrX:33371523-33371643,chrX:33372317-33372437,chrX:33373358-33373478,chrX:33374562-33374682,chrX:33375515-33375635,chrX:33376317-33376437,chrX:33377656-33377776；
group II:
chrX:31139471-31139591,chrX:31140475-31140595,chrX:31144475-31144595,chrX:31152501-31152621,chrX:31164474-31164594,chrX:31165472-31165592,chrX:31187475-31187595,chrX:31190600-31190720,chrX:31191470-31191590,chrX:31195475-31195595,chrX:31196665-31196785,chrX:31198475-31198595,chrX:31200475-31200595,chrX:31222424-31222544,chrX:31224664-31224784,chrX:31227475-31227595,chrX:31241464-31241584,chrX:31279439-31279559,chrX:31284475-31284595,chrX:31341389-31341509,chrX:31366615-31366735,chrX:31462541-31462661,chrX:31496444-31496564,chrX:31497425-31497545,chrX:31514499-31514619,chrX:31525475-31525595,chrX:31526475-31526595,chrX:31645497-31645617,chrX:31676453-31676573,chrX:31697475-31697595,chrX:31747433-31747553,chrX:31792530-31792650,chrX:31838515-31838635,chrX:31854489-31854609,chrX:31893384-31893504,chrX:31947527-31947647,chrX:31950484-31950604,chrX:31986475-31986595,chrX:32173475-32173595,chrX:32234629-32234749,chrX:32305613-32305733,chrX:32328289-32328409,chrX:32360417-32360537,chrX:32361314-32361434,chrX:32364410-32364530,chrX:32366521-32366641,chrX:32380562-32380682,chrX:32382475-32382595,chrX:32383642-32383762,chrX:32398600-32398720,chrX:32404459-32404579,chrX:32407540-32407660,chrX:32408363-32408483,chrX:32429493-32429613,chrX:32430393-32430513,chrX:32456417-32456537,chrX:32459498-32459618,chrX:32466496-32466616,chrX:32472449-32472569,chrX:32481475-32481595,chrX:32482475-32482595,chrX:32486585-32486705,chrX:32490356-32490476,chrX:32503475-32503595,chrX:32509475-32509595,chrX:32519584-32519704,chrX:32536576-32536696,chrX:32563461-32563581,chrX:32583475-32583595,chrX:32591548-32591668,chrX:32592587-32592707,chrX:32601531-32601651,chrX:32613409-32613529,chrX:32632444-32632564,chrX:32659367-32659487,chrX:32662386-32662506,chrX:32663657-32663777,chrX:32716475-32716595,chrX:32717501-32717621,chrX:32827561-32827681,chrX:32834528-32834648,chrX:32841482-32841602,chrX:32862555-32862675,chrX:32868323-32868443,chrX:33038429-33038549,chrX:33146475-33146595,chrX:33229510-33229630,chrX:33357475-33357595；
group III:
chrX:31645497-31645617,chrX:31676453-31676573,chrX:31697475-31697595,chrX:31747433-31747553,chrX:31792530-31792650,chrX:31838515-31838635,chrX:31854489-31854609,chrX:31893384-31893504,chrX:31947527-31947647,chrX:31950484-31950604,chrX:31986475-31986595,chrX:32519584-32519704,chrX:32536576-32536696,chrX:32563461-32563581,chrX:32583475-32583595,chrX:32591548-32591668,chrX:32592587-32592707,chrX:32601531-32601651,chrX:32613409-32613529,chrX:32632444-32632564,chrX:32659367-32659487,chrX:32662386-32662506,chrX:32663657-32663777,chrX:32716475-32716595,chrX:32717501-32717621,chrX:32827561-32827681,chrX:32834528-32834648,chrX:32841482-32841602,chrX:32862555-32862675,chrX:32868323-32868443,chrX:33038429-33038549,chrX:33146475-33146595,chrX:33229510-33229630。
wherein the chromosomal region is a region of the human Dystrophin gene (or DMD gene), and the reference sequence is human reference genome version Hg19(chrX: 31137344-33357726).
1. Sample genomic DNA extraction and quality assessment
Taking 0.5-1 m L of peripheral Blood of a sample to be detected, adopting Blood Genomic DNA Mini Kit (Beijing kang is century Biotechnology Co., Ltd., peripheral Blood) to extract genome DNA, detecting the integrity of the genome through agarose gel electrophoresis, using Qubit (L if Technologies, USA) to carry out concentration determination on the genome, and selecting a DNA sample with relatively complete genome and the concentration of more than or equal to 25 ng/microliter to construct a library.
2. Construction of the Capture library
2.1 disruption of genomic DNA
The method comprises the following steps of (1) breaking genomic DNA by using a Covaris E220 non-contact ultrasonic breaker, and setting parameters according to the average length of genome fragmentation of 5K, wherein the parameters are as follows: duration 600sec, Peak incorporated Power 3 Watts, Duty Factor 20%, Cycles per Burst 1000.
The purification was performed using 0.6-fold volume of AMPure PB magnetic beads to obtain fragmented genomic DNA.
2.2 sample end repair, Add Joint
2.2.1 end repair, adding "A"
The NEB Next Ultra End Repair/dA-labeling Module kit was used.
The system is shown in table 1 below:
TABLE 1
Figure BDA0002555845440000231
Figure BDA0002555845440000241
The reaction conditions are shown in table 2 below:
TABLE 2
Figure BDA0002555845440000242
2.2.2 Joint connection
NEB Next Ultra II L alignment Module kit was used.
The system is shown in table 3 below:
TABLE 3
Figure BDA0002555845440000243
The reaction was carried out in a PCR apparatus at 20 ℃ for 15 min. Subsequently, purification was performed using 0.6-fold volume of AMPure XP magnetic beads to obtain a library solution.
Wherein, the joint is composed of an A sequence and a B sequence, the A sequence is:
5 '-pGATCGGAAGCACGTCTGAACTNNNNNNNNNNNNAGGGACGTGCGCCATAGAAG-3' (SEQ ID NO: 1); wherein N is any one of A, C, T or G;
the sequence B is:
5'-CTTCCAGAAGAGGCCATAGCAGATNNNNNNNNNNNNNNNNAGTTCAGACGTGTGCTCTTCCGATCT/3SpC3/-3' (SEQ ID NO: 2); wherein N is any one of A, C, T or G; p in the above sequence represents a phosphorylation modification; n represents any one of four bases of A/G/C/T; /3SpC 3/represents a phosphorothioate linkage modification; sequence a nnnnnnnnnnnn and sequence B NNNNNNNNNNNNNNNN were used to identify libraries constructed from different samples.
2.3 Pre-amplification of the library, the reaction system is shown in Table 4 below:
TABLE 4
Figure BDA0002555845440000244
Figure BDA0002555845440000251
Wherein, the Primer is a Primer for library pre-amplification, and specifically comprises the following components:
Primer F：5'-CTTCTATGGCGCACGTCCCT-3'(SEQ ID NO:5)，
Primer-R：5'-CTTCCAGAAGAGGCCATAGCA-3'(SEQ ID NO:6)。
the reaction conditions are shown in table 5 below:
TABLE 5
Figure BDA0002555845440000252
PCR products were recovered using 0.6 volumes of AMPure XP magnetic beads.
2.4 hybrid Capture target sequences
2.4.1 Capture preparation
The samples to be captured were mixed in equal amounts, the total amount of DNA being 1. mu.g.
Add 4 u L Index blocking reagent (Index A Block, Index B Block), its purpose is to Block the library construction used in the joint A sequence and B sequence, respectively, prevent the library of the two joint sequences and probe hybridization.
The Index A Block sequence is:
5 '-CTTCTATGGCGCACGTCCCTNNNNNNNNNNAGTTCAGACGTGTGCTCTTCCGATC-3' (SEQ ID NO: 3); wherein N is any one of A, C, T or G;
the Index B Block sequence is:
5 '-AGATCGGAAGAGCACACGTCTGAACTNNNNNNNNNNNNNNNNATCTGCTATGGCCTCTTCTGGAAG-3' (SEQ ID NO: 4); wherein N is any one of A, C, T or G.
N in the sequence represents any one of four bases of A/G/C/T, wherein Index A Block is reversely complementary with the sequence of the adaptor A; index B Block is reverse complementary to the linker B sequence.
Shaking and mixing evenly, then opening a cover and vacuum concentrating to dryness at 60 ℃.
2.4.2 Capture
The hybridization buffer was prepared according to the instruction of the Kit XGen L ockdown Reagent Kit (IDT, cat # 1072281), and the specific system is shown in the following Table 6:
TABLE 6
Figure BDA0002555845440000253
Adding the hybridization buffer into the evaporated sample, shaking and uniformly mixing, centrifuging for a short time, and placing on a PCR instrument at 95 ℃ for 10 min.
After the reaction was completed, the reaction mixture was centrifuged, and the 13. mu. L sample was transferred to a PCR tube containing a capture probe at a concentration of 0.75pmol/ul in a volume of 4. mu. L, and the mixture was pipetted and mixed, and the mixture was placed on a PCR instrument at 65 ℃ for 16 hours.
2.5 hybridization elution
2.5.1 elution reagents were formulated according to the instructions for the Kit XGen L ockdown Reagent Kit (IDT, cat # 1072281) as shown in Table 7 below:
TABLE 7
Figure BDA0002555845440000261
2.5.2 Capture magnetic bead treatment
a) 50 mu l M280 magnetic beads were pipetted into the centrifuge tube, the centrifuge tube was placed on a magnetic rack until the supernatant was completely clarified, and the supernatant was discarded.
b) Add 200. mu.l of 1 × Bead Wash Buffer (via 7), mix well, centrifuge, place on magnetic rack until the supernatant is clear, discard the supernatant.
c) Repeating the step b once.
d) Add 100. mu.l of 1 × Bead Wash Buffer (via 7), mix well, transfer to a PCR tube, place on a magnetic rack until the supernatant is completely clear, and discard the supernatant.
2.5.3 binding of magnetic beads to target libraries
Add 17. mu.l of the hybridization system to the PCR tube with magnetic beads, blow and suck up slowly and mix them well, incubate them for 45min at 65 ℃ on the PCR instrument.
2.5.4 elution
a) 100 μ l of 1 × Wash Buffer I (visual 1) at 65 ℃ was added, mixed well, placed on a magnetic stand until the supernatant was completely clarified, and the supernatant was discarded.
b) 200 μ l of 65 ℃ 1 × Stringent Wash Buffer (via 4) was added, mixed well and transferred to a large pre-heated centrifuge tube, placed on an incubator at 65 ℃ for 5min, placed on a magnetic rack and the supernatant discarded after it was completely clarified.
c) Adding 200 μ l of normal temperature 1 × Wash Buffer I (visual 1), mixing, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
d) Adding 200 μ l of normal temperature 1 × Wash Buffer II (visual 2), mixing uniformly 1, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
e) Adding 200 μ l of normal temperature 1 × Wash Buffer III (visual 3), mixing, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
f) Briefly centrifuged and a small amount of remaining liquid was removed using a 10. mu.l tip.
g) The magnetic beads were suspended in 25. mu.l of nucleic-free Water and stored at-20 ℃.
2.6 hybrid library amplification
The M280 magnetic bead probe suspension obtained in the previous step is prepared by taking 25 mu L/library and performing a PCR amplification system in a 200ml PCR tube according to the following table 8:
TABLE 8
Figure BDA0002555845440000262
Figure BDA0002555845440000271
The primers described above are consistent with those pre-amplified from the library above:
Primer F：5'-CTTCTATGGCGCACGTCCCT-3'(SEQ ID NO:5)，
Primer-R：5'-CTTCCAGAAGAGGCCATAGCA-3'(SEQ ID NO:6)。
the PCR reaction procedure is shown in table 9 below:
TABLE 9
Figure BDA0002555845440000272
After the reaction is finished, AMPure XP magnetic beads with the volume of 0.6 time are used for purification, and finally TE solution is used for eluting the magnetic beads, and the magnetic beads are transferred into a new centrifugal tube to enter the next operation or placed at-80 ℃ for standby.
2.7 library quality testing
The library is subjected to Qbuit detection and Agilent 2100Bioanalyzer detection, and a qualified library is detected to be subjected to next generation sequencing library construction.
3. Third generation sequencing library construction and sequencing
3.1 sequencing Using Pacbio sequal
3.1.1 library construction Using library construction kit
3.1.1.1 repair of pooled libraries
The repair solutions were formulated as shown in table 10 below:
watch 10
Figure BDA0002555845440000273
Mixing, centrifuging, and placing into a PCR thermal cycler for repair reaction under the following specific conditions:
TABLE 11
Figure BDA0002555845440000274
Figure BDA0002555845440000281
3.1.1.2 purification
Purification was performed using 0.45 sample volumes of PB magnetic beads, and finally the beads were eluted using double distilled water and stored in a-20 ℃ freezer.
3.1.1.3 Joint connections
The bonding solution system is shown in table 12 below:
TABLE 12
Figure BDA0002555845440000282
Mixing, centrifuging, and performing ligation reaction in a PCR thermal cycler under the following specific conditions:
watch 13
Figure BDA0002555845440000283
3.1.1.4 purification
Purification was performed using 0.45 sample volumes of PB magnetic beads, and finally the beads were eluted using double distilled water and stored in a-20 ℃ freezer.
3.1.2 primer annealing and Binding reactions
The procedure was performed in the standard manner of operation given by the Pacbio official webpage.
3.1.3 third Generation sequencing
Sequencing was performed according to the standard operating specifications of the Pacbio sequal instrument.
3.2 sequencing Using Oxford Nanopore PromethION
3.2.1 sample end repair
DNA is taken and placed on ice, NEB end repairing/A tail reagent is added, and the mixture is mixed evenly. Incubate at 20 ℃ for 40 minutes and at 65 ℃ for 20 minutes.
3.2.2 DNA purification
1 × AMPure beads were added to the DNA, incubated for 15min at room temperature, magnetically mounted for 5min at room temperature, and the supernatant was discarded.
Adding 80% ethanol, adsorbing with magnetic frame, discarding supernatant, and repeating once. And (5) drying at room temperature.
Ultra Pure Water was added and the elution was performed by pipetting at 37 ℃.
Standing for 5min on a magnetic frame, and sucking supernatant to obtain purified DNA.
3.2.3 Joint connection
Adding NEB T4 DNA fast connection buffer, NEB T4 DNA fast ligase and adaptor, mixing, and incubating at 20 deg.C for 20 min.
3.2.4 DNA purification
Same step 3.2.2
3.2.5 primer annealing and L loading reactions
The operation is carried out according to the standard operation mode given by the Oxford Nanopore PromethION official webpage.
3.2.6 third Generation sequencing
Sequencing was performed according to Oxford Nanopore PromethION Instrument Standard protocol.
Bioinformatics analysis
4.1 splitting the data for each sample
And if the off-line data is multi-sample mixed measurement, splitting the data of each sample according to the label sequence of each sample.
4.2 data Filtering
And generating high-quality data according to the data characteristics of each platform.
The ONT platform filters out data with lower quality values. The Pacbio platform employs official software to generate high quality consensus sequences.
4.3 data evaluation
High quality data were aligned to human whole genome reference sequences and unique alignments were taken (i.e., a single sequence could only be aligned to one position of the genome).
And counting the sequence number of the target area, and evaluating the capture efficiency, the coverage depth of the target area and the coverage integrity. If the data is not qualified (the qualified standard is that the sequencing coverage of the target region requires that the coverage is not less than 95 percent by 30 times), the data is subjected to additional measurement according to the situation.
4.4 detection of variation
Detecting single nucleotide site variation, small fragment insertion and deletion, chromosome structure variation (SNV/InDel, SV) and the like of a target region; and annotating the variation with each database to screen for highly pathogenic variations.
4.5 validation of the variation.
And carrying out first-generation verification on the screened highly pathogenic mutation.
The M L PA can only detect the deletion and the duplication of the DMD gene exon, can not detect the tiny deletion, duplication and point mutation, the detection rate is about 60 percent, and obvious missing detection exists, although Sanger sequencing can detect the mutation, inversion, ectopy, tiny duplication and deletion mutation, but can not detect the large fragment duplication, particularly, in view of low sequencing flux of Sanger, three thousand sequencing reactions need to be carried out for realizing the whole gene sequencing of the DMD of the ultralong gene, the cost is high, the workload is large, therefore, clinically, Sanger only serves as a means for further accurately verifying the mutation found in the results of the second-generation sequencing, and the M L is a region containing the tiny duplication of less than 20 bp.
The disease condition of the DMD is confirmed by observing clinical manifestations and muscle case biopsy of a plurality of collected samples, and meanwhile, the samples are detected by using the method, M L PA, Sanger sequencing, second-generation capture sequencing and a method combining M L PA and second-generation capture sequencing.
The results of the tests performed on 37 samples according to the protocol of the present invention are shown in Table 14 below, where "EX" represents exon, "c." represents cDNA, "p." represents protein, "DE L" represents deletion type variation, "DUP" represents repeat type variation, "INV" represents inversion type variation, "TRA" represents translocation type variation, "het" represents heterozygote, and "hemizygote.
TABLE 14
Figure BDA0002555845440000301
Figure BDA0002555845440000311
9. No. 21 and No. 32 samples, which are 2 healthy human standard samples and 1 healthy human sample, are not detected with actual conditions, No. 28 and No. 37 samples are detected with pathogenic variable sites, but the second-generation sequencing result shows that no pathogenic variable sites are detected, furthermore, the two samples are verified by using the first-generation sequencing result, the first-generation sequencing result shows that the first-generation sequencing result is consistent with the result, and clinical observation and muscle case biopsy show that the pathogenic variable sites are typical shown, the other samples are all consistent with the second-generation sequencing result, all pathogenic variable sites are detected, and the types and sites of the pathogenic variable sites are consistent and have the clinical display of DMD, in addition, the samples of the detected DUP and DE L variant types are simultaneously verified by an M L PA experiment, and the verification results also show that the samples are all consistent:
1. the invention can detect the structural variation of large segment repetition, inversion and ectopic types; such as sample numbers 28, 37 above;
2. the detection rate of the invention is higher than that of other technical means; the method of the invention realizes the complete detection of the pathogenic variable sites of 35 samples with clinical manifestations;
3. the invention can detect a large number of mutations and provides a molecular biological basis for discovering new pathogenic mutations.
Example 2
TSCl and TSC2 are both oncogenes that exert negative regulatory effects on cell proliferation and differentiation, and are typically characterized by benign tumors that involve all other systems and organs except the peripheral nerve, skeletal muscle, and pinecone. Mutations or deletions of the TSC1 and TSC2 genes can cause Tuberous Sclerosis (TSC), also known as Bourneville disease, which is a clinically common autosomal dominant hereditary neurocutaneous syndrome. The incidence rate is 1/10000-1/6000, 60% -70% of sporadic cases have no family history and show high spontaneous mutation rate. The ratio of the male and female diseases is 2:1, and no obvious ethnic difference exists. The research shows that the TSC1 and TSC2 gene abnormality causes the abnormality of the functions of the transcription products, namely hamartoma protein and potato protein, so that the normal processes of cell generation, differentiation and migration are influenced, and the pathological condition mainly shows that the abnormal shape, number, position and structural arrangement of cells form tumor-like lesion. The TSC2 mutation accounted for 3/5 while the TSC1 mutation accounted for 3/10 in TSC patients, and some clinically confirmed patients did not detect the mutation, possibly located in an undetected non-coding region (UTR), or a regulatory region distant from the coding exon, etc. Meanwhile, there is a certain difference between mutant phenotypes of TSC1 and TSC2: overall, patients with variations in TSC2 had more severe clinical manifestations, more involvement of the kidney and reduced intelligence, and more pronounced brain and facial damage. The TSC2 variant had renal cysts earlier in age than the TSC1 variant, with numerous cysts and large volume. In addition, all TSC patients with polycystic kidneys had mutations in TSC2 gene, the TSC2 gene was only 60bp from the polycystic kidney gene (PKD1), and gene analysis found that 90% of these patients had simultaneous deletion of TSC2 and the adjacent PKD1 gene, and 10% had other types of mutations in TSC2 and PKD1 genes, suggesting that deletion of TSC2 may affect neighboring genes, resulting in the syndrome of deletion of the vicinal (contigous) gene.
The method is used for determining the average interval range of the probes for TSC1 and TSC2, the average genome fragmentation length L is set to be 5Kb, the average length P of each probe is set to be 100bp, the average length P is substituted into a formula N (L +2) × P +/-3 × P, the average probe interval N is calculated to be 400bp-1000 bp., 500bp is selected to be the average probe interval in the range, in the design process, the GC content value of the probes is ensured to be moderate, no hairpin structure is required, Blast comparison is further carried out with databases such as NCBI, the probes are ensured to avoid the repeated region on the genome, the specificity of the probes is ensured, and finally determined probe sequences are shown in the information of TSC1 and TSC2 probes.
TSC1 and TSC2 probe cases are shown as "TSC probe set".
1. Sample genomic DNA extraction and quality assessment
Taking 0.5-1 m L of peripheral Blood of a sample to be detected, adopting Blood Genomic DNA Mini Kit (Beijing kang is century Biotechnology Co., Ltd., peripheral Blood) to extract genome DNA, detecting the integrity of the genome through agarose gel electrophoresis, using Qubit (L if Technologies, USA) to carry out concentration determination on the genome, and selecting a DNA sample with relatively complete genome and the concentration of more than or equal to 25 ng/microliter to construct a library.
2. Construction of the Capture library
2.1 disruption of genomic DNA
Fragmented genomic DNA was prepared by disrupting genomic DNA using Covaris G-TUBE (10)520079E220 centrifugation at high speed with the parameters set forth in Gene Company L impacted centrifuge 1524 at 20000G rpm for 30S, a mass of 1-1.5ug of gDNA, a volume of 100ul, and repeated three times (4-6 disruptions).
2.2 sample end repair, Add Joint
2.2.1 end repair, adding "A"
The NEB Next Ultra End Repair/dA-labeling Module kit was used.
The system is shown in table 15:
watch 15
Figure BDA0002555845440000321
The reaction conditions are shown in table 16:
TABLE 16
Figure BDA0002555845440000322
2.2.2 Joint connection
NEB Next Ultra II L alignment Module kit was used.
The system is shown in table 17:
TABLE 17
Figure BDA0002555845440000323
Reaction second step linker ligation reaction in PCR Instrument (or overnight ligation at 4 ℃) the conditions are shown in Table 18:
watch 18
Figure BDA0002555845440000331
Subsequently, purification was performed using 0.5-fold volume of AMPure XP magnetic beads, and library solution conditions were obtained as shown in table 19.
2.3 Pre-amplification of the library, the reaction system is shown in Table 19:
watch 19
Figure BDA0002555845440000332
Wherein, the Primer is a Primer for library pre-amplification, and specifically comprises the following components:
Primer F：5'-CTTCTATGGCGCACGTCCCT-3'(SEQ ID NO:5)，
Primer-R：5'-CTTCCAGAAGAGGCCATAGCA-3'(SEQ ID NO:6)。
the reaction conditions are shown in table 20 below:
watch 20
Figure BDA0002555845440000333
PCR products were recovered using 0.5 volumes of AMPure PB magnetic beads.
2.4 hybrid Capture target sequences
2.4.1 Capture preparation
The samples to be captured were mixed in equal amounts, the total amount of DNA being 1. mu.g.
Add 4 u L Index blocking reagent (Index A Block, Index B Block), its purpose is to Block the library construction used in the joint A sequence and B sequence, respectively, prevent the library of the two joint sequences and probe hybridization.
The Index A Block sequence is:
5'-CTTCTATGGCGCACGTCCCTNNNNNNNNNNAGTTCAGACGTGTGCTCTTCCGATC–3'(SEQ IDNO:3)；
the Index B Block sequence is:
5'-AGATCGGAAGAGCACACGTCTGAACTNNNNNNNNNNNNNNNNATCTGCTATGGCCTCTTCTGGAAG–3'(SEQ ID NO:4)。
n in the sequence represents any one of four bases of A/G/C/T, wherein Index A Block is reversely complementary with the sequence of the adaptor A; index B Block is reverse complementary to the linker B sequence.
Shaking and mixing evenly, then opening a cover and vacuum concentrating to dryness at 60 ℃.
2.4.2 Capture
The hybridization buffer was prepared according to the instruction of the Kit XGen L ockdown Reagent Kit (IDT, cat # 1072281), and the specific system is shown in Table 21:
TABLE 21
Figure BDA0002555845440000341
Adding the hybridization buffer into the evaporated sample, shaking and uniformly mixing, centrifuging for a short time, and placing on a PCR instrument at 95 ℃ for 10 min.
After the reaction, the reaction mixture was centrifuged, and the 13. mu. L sample was transferred to a PCR tube containing 4. mu. L capture probe, and the mixture was pipetted and mixed well on a PCR instrument at 65 ℃ for 16 hours.
2.5 hybridization elution
2.5.1 elution reagents were formulated according to the instructions of Kit XGen L ockdown Reagent Kit (IDT, cat # 1072281) as shown in Table 22:
TABLE 22
Figure BDA0002555845440000342
2.5.2 Capture magnetic bead treatment
a) 50 mu l M280 magnetic beads were pipetted into the centrifuge tube, the centrifuge tube was placed on a magnetic rack until the supernatant was completely clarified, and the supernatant was discarded.
b) Add 200. mu.l of 1 × Bead Wash Buffer (via 7), mix well, centrifuge, place on magnetic rack until the supernatant is clear, discard the supernatant.
c) Repeating the step b once.
d) Add 100. mu.l of 1 × Bead Wash Buffer (via 7), mix well, transfer to a PCR tube, place on a magnetic rack until the supernatant is completely clear, and discard the supernatant.
2.5.3 binding of magnetic beads to target libraries
Add 17. mu.l of the hybridization system to the PCR tube with magnetic beads, blow and suck up slowly and mix them well, incubate them for 45min at 65 ℃ on the PCR instrument.
2.5.4 elution
a) 100 μ l of 1 × Wash Buffer I (visual 1) at 65 ℃ was added, mixed well, placed on a magnetic stand until the supernatant was completely clarified, and the supernatant was discarded.
b) 200 μ l of 65 ℃ 1 × Stringent Wash Buffer (via 4) was added, mixed well and transferred to a large pre-heated centrifuge tube, placed on an incubator at 65 ℃ for 5min, placed on a magnetic rack and the supernatant discarded after it was completely clarified.
c) Adding 200 μ l of normal temperature 1 × Wash Buffer I (visual 1), mixing, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
d) Adding 200 μ l of normal temperature 1 × Wash Buffer II (visual 2), mixing uniformly 1, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
e) Adding 200 μ l of normal temperature 1 × Wash Buffer III (visual 3), mixing, placing on a magnetic rack, and discarding the supernatant when the supernatant is completely clear.
f) Briefly centrifuged and a small amount of remaining liquid was removed using a 10. mu.l tip.
g) The magnetic beads were suspended in 25. mu.l of nucleic-free Water and stored at-20 ℃.
2.6 hybrid library amplification
The M280 magnetic bead probe suspension obtained in the previous step was prepared by taking 25. mu. L/library and performing a PCR amplification system in a 200ml PCR tube according to the following Table 23:
TABLE 23
Figure BDA0002555845440000351
The primers described above are consistent with those pre-amplified from the library above:
Primer F：5'-CTTCTATGGCGCACGTCCCT-3'(SEQ ID NO:5)，
Primer-R：5'-CTTCCAGAAGAGGCCATAGCA-3'(SEQ ID NO:6)。
the PCR reaction program is shown in table 24:
watch 24
Figure BDA0002555845440000352
After the reaction is finished, AMPure XP magnetic beads with the volume of 0.6 time are used for purification, and finally TE solution is used for eluting the magnetic beads, and the magnetic beads are transferred into a new centrifugal tube to enter the next operation or placed at-80 ℃ for standby.
2.7 library quality testing
The library is subjected to Qbuit detection and Agilent 2100Bioanalyzer detection, and a qualified library is detected to be subjected to next generation sequencing library construction.
3. Third generation sequencing library construction and sequencing
3.1 sequencing Using Pacbio sequal
3.1.1 library construction Using library construction kit
3.1.1.1 repair of pooled libraries
The repair solutions were formulated as shown in table 25:
TABLE 25
Figure BDA0002555845440000361
Mixing, centrifuging, and placing into a PCR thermal cycler for repair reaction under the following specific conditions:
watch 26
Figure BDA0002555845440000362
3.1.1.2 purification
Purification was performed using 0.45 sample volumes of PB magnetic beads, and finally the beads were eluted using double distilled water and stored in a-20 ℃ freezer.
3.1.1.3 Joint connections
The bonding solution system is shown in table 27 below:
watch 27
Figure BDA0002555845440000363
Mixing, centrifuging, and performing ligation reaction in a PCR thermal cycler under the following specific conditions:
watch 28
Figure BDA0002555845440000364
3.1.1.4 purification
Purification was performed using 0.45 sample volumes of PB magnetic beads, and finally the beads were eluted using double distilled water and stored in a-20 ℃ freezer.
3.1.2 primer annealing and Binding reactions
The procedure was performed in the standard manner of operation given by the Pacbio official webpage.
3.1.3 third Generation sequencing
Sequencing was performed according to the standard operating specifications of the Pacbio sequal instrument.
3.2 sequencing Using Oxford Nanopore PromethION
3.2.1 sample end repair
DNA is taken and placed on ice, NEB end repairing/A tail reagent is added, and the mixture is mixed evenly. Incubate at 20 ℃ for 40 minutes and at 65 ℃ for 20 minutes.
3.2.2 DNA purification
1 × AMPure beads were added to the DNA, incubated for 15min at room temperature, magnetically mounted for 5min at room temperature, and the supernatant was discarded.
Adding 80% ethanol, adsorbing with magnetic frame, discarding supernatant, and repeating once. And (5) drying at room temperature.
Ultra Pure Water was added and the elution was performed by pipetting at 37 ℃.
Standing for 5min on a magnetic frame, and sucking supernatant to obtain purified DNA.
3.2.3 Joint connection
Adding NEB T4 DNA fast connection buffer, NEB T4 DNA fast ligase and adaptor, mixing, and incubating at 20 deg.C for 20 min.
3.2.4 DNA purification
Same step 3.2.2
3.2.5 primer annealing and L loading reactions
The operation is carried out according to the standard operation mode given by the Oxford Nanopore PromethION official webpage.
3.2.6 third Generation sequencing
Sequencing was performed according to Oxford Nanopore PromethION Instrument Standard protocol.
Bioinformatics analysis
4.1 splitting the data for each sample
And if the off-line data is multi-sample mixed measurement, splitting the data of each sample according to the label sequence of each sample.
4.2 data Filtering
And generating high-quality data according to the data characteristics of each platform.
The ONT platform filters out data with lower quality values. The Pacbio platform employs official software to generate high quality consensus sequences.
4.3 data evaluation
High quality data were aligned to human whole genome reference sequences and unique alignments were taken (i.e., a single sequence could only be aligned to one position of the genome).
And counting the sequence number of the target area, and evaluating the capture efficiency, the coverage depth of the target area and the coverage integrity. If the data is not qualified (the qualified standard is that the sequencing coverage of the target region requires that the coverage is not less than 95 percent by 30 times), the data is subjected to additional measurement according to the situation.
4.4 detection of variation
Detecting single nucleotide site variation, small fragment insertion and deletion, chromosome structure variation (SNV/InDel, SV) and the like of a target region; and annotating the variation with each database to screen for highly pathogenic variations.
4.5 validation of the variation.
And carrying out first-generation verification on the screened highly pathogenic mutation.
The method is only suitable for detecting the deletion or rearrangement of large TSC gene fragments and needs to be combined with other detection methods, so that the mutation detection of TSC1 gene with large fragment gene deletion or gene rearrangement is not suitable, long-short polymerase chain reaction-single strand polymorphism analysis (PCR-SSCP) can improve the detection rate of the mutation to a greater extent, but has certain limitation on the deletion or rearrangement of large fragments, the sensitivity is lower than that of other methods, the operation is time-consuming and labor-consuming, the sequencing of the mutation can be greatly improved, the detection sensitivity is very high for the deletion or rearrangement of large fragments, the detection cost is very high for detecting the deletion or rearrangement of special TSC gene fragments, the detection cost is very low, the detection cost is very high for detecting the deletion or rearrangement of special TSC gene fragments, the detection cost is only low for detecting the deletion or rearrangement of TSC gene fragments by using a standard sequencing-based on the TSC-DNA sequencing technology, the detection method is only a high-600-long sequencing probe, the detection cost is very low for detecting the deletion or rearrangement of the mutation of the TSC gene, the TSC gene mutation is very high-20-long sequencing gene deletion or the TSC gene rearrangement, the TSC gene mutation detection of the TSC gene mutation detection of the mutation, the mutation detection of the mutation of the TSC gene mutation of the TSC gene deletion or the TSC gene, the TSC gene mutation of the TSC gene, the TSC gene of the TSC gene is very high-DNA, the TSC gene of the TSC gene, the TSC gene of the TSC gene, the TSC gene of the TSC gene.
The micro-repeat means that a repeat region is smaller than 20bp, and the large-fragment repeat means that the repeat region is larger than 20 bp. and the M L PA and the TSC2 gene exon region only aim at the TSC1 and TSC2 gene exon region, if the sequence is carried out on an intron, the whole detection cost is increased by more than 100 times, and high detection cost cannot bear patients.
Meanwhile, the samples are detected by using the method, the M L PA, Sanger sequencing, second-generation capture sequencing and the combined use of the M L PA and the second-generation capture sequencing.
The results show that:
1. the invention can detect the structural variation of large segment repetition, inversion and ectopic types;
2. the detection rate of the invention is higher than that of other technical means;
3. the invention can detect a large number of mutations and provides a molecular biological basis for discovering new pathogenic mutations.
Example 3
Hybridization Probe effective Interval Length test
The percentage of sequences with an average depth of coverage of 0.5 × is a relatively common parameter for assessing capture uniformity and is used to examine the limitations associated with low depth of coverage. if more than 90% of the target area exhibits an average depth of coverage exceeding 0.5 ×, then Panel's uniformity of coverage is better.
Taking the DMD gene capture described in example 1 as an example, in the "2.1 disruption of genomic DNA" step in example 1, by setting different parameters, fragmentation of different lengths of genomic DNA was achieved, fragmentation of 2Kb and 5Kb was performed using ultrasound as in example 1, fragmentation of 8Kb was performed using centrifugation, and the fragmentation size and specific parameters were as shown in tables 29 and 30 below:
watch 29
Figure BDA0002555845440000391
Watch 30
Figure BDA0002555845440000392
The average length P of each probe is 120bp, and for each fragmented size, the influence of the interval distance of 0bp (N ═ 0P), 1 bp (N ═ 1P), 4 bp (N ═ 4P), 840bp (N ═ 7P) was measured, until 19 bp (N ═ 19P) was measured, on the uniformity of coverage of the target region. The specific experimental procedure is as described in example 1.
Watch 31
Figure BDA0002555845440000393
Capture DNA fragments with an average length of 2Kb for genomic fragmentation:
the specific numerical values of the Pacbio platform test results are shown in the table 31, the average coverage depth ratio of 0.5 × is shown in fig. 2, the average distance between 0P and 1P has almost no influence on the coverage uniformity of the target region, the average distance between 1P and 7P has a weak influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a weak downward trend but the ratio still exceeds 90%, the distance above the average interval 7P has a significant influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a significant downward trend and the ratio is lower than 90%.
Watch 32
Figure BDA0002555845440000401
Capture DNA fragments with an average length of 5Kb for genomic fragmentation:
the specific values of the test results of the PacBio platform are shown in the table 32, the average coverage depth ratio of 0.5 × is shown in fig. 1, the average distance between 0P and 4P has almost no influence on the coverage uniformity of the target region, the average distance between 4P and 10P has a weak influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a weak downward trend but the ratio still exceeds 90%, the distance above the average interval 10P has a significant influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a significant downward trend and the ratio is lower than 90%, therefore, the average interval length N of the hybridization probe is preferably controlled within 10P for a capture DNA fragment with the average genome fragmentation length of 5Kb, and the average interval range of the probe obtained by calculation of (L +2) × P +/-3 × P is 4P to 10P and is consistent with the test results.
Watch 33
Figure BDA0002555845440000402
Capture DNA fragments with an average length of 8Kb for genomic fragmentation:
the specific values of the test results of the PacBio platform are shown in the table 33, the average coverage depth ratio of 0.5 × is shown in fig. 3, the average distance between 0P and 7P has almost no influence on the coverage uniformity of the target region, the average distance between 7P and 13P has a weak influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a weak downward trend but the ratio still exceeds 90%, the distance above the average interval 13P has a significant influence on the coverage uniformity of the target region, the average coverage depth target region ratio of 0.5 × has a significant downward trend and the ratio is lower than 90%, therefore, the effective interval length N of the hybridization probe is preferably controlled within 13P for a capture DNA fragment with the average genome fragmentation length of 8Kb, and the average interval range of the probe, namely 7P to 13P calculated by a formula (L +2) × P +/-3 × P, is consistent with the test results.
Example 4
Probe addition and target in Capture test
The target-in-capture rate of the target fragment is closely related to the concentration of the capture probe, wherein the target-in-capture rate refers to the ratio of the number of bases of the target fragment to the total number of bases of the next machine in the capture result. For this purpose, the relationship between the amount of capture probes added and the target rate in capturing the target fragment was examined, with an average interval of 7 probes (N-7P).
Using the DMD gene capture described in example 1 as an example, the relationship between the amount of capture probe added and the target rate in capture of the target fragment was tested. In example 1, the average length of the probes was 120bp, and the average interval between the probes was 1 Kb. Specifically, in steps 1 and 2.4.2, a gradient of the amount of probe added was set (3, 0.3, 0.03, 0.003), and the other steps were performed as in example 1, and the results were measured using the PacBio platform, and the specific data are shown in table 34 below:
watch 34
Figure BDA0002555845440000411
As shown in FIG. 4, the target rate in the capture of the target fragment gradually decreases as the amount of the capture probe to be added decreases. Therefore, in order to achieve a target-in-capture ratio of 50% or more, the amount of the capture probe with N-7P added should be controlled to 0.3pmol or more.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. The preparation method of the long fragment capture sequencing probe set is characterized by comprising the following steps:
a) the average probe spacing range was calculated using the following formula:
N＝(L+2)×P±3×P；
wherein:
n: average probe spacing in bp;
p: average length of each probe, unit bp;
l genome fragmentation average length unit Kb, taking an integer;
b) preparing a probe set for detecting the target region according to the average interval range of the probes.
2. The method of claim 1, wherein the value of P ranges from 15bp to 250 bp; preferably 100bp to 200 bp.
3. The method of claim 1, wherein the value of L is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10.
4. The method of claim 1, wherein the species of the target region is of human origin.
5. The method of claim 1, further comprising grouping the probe sets, the grouping selected from at least one of the following groups:
(1) capturing the probe for the full length of the target region;
(2) a probe that captures only an exon region of the target region;
(3) probes that capture only the high mutation rate region;
preferably, the method further comprises coupling the probe to a label bound to a solid phase, the label being selected from one or more of streptavidin, biotin, avidin, an antibody, a chemical coupling agent.
6. The method of any one of claims 1 to 5, wherein the target region comprises:
any one of a dyetropin gene, a TSC1 gene, a TSC2 gene, a BRAC1 gene, a BRAC2 gene, a GJB2 gene, a 12SrRNA gene, or a S L C26a4 gene.
7. Use of the method of any one of claims 1 to 6 for third generation sequencing;
preferably, the third generation sequencing is performed using a PacBio sequence, PromethION, MinION or GridION platform.
8. A probe set prepared by the method of any one of claims 1 to 6;
optionally, the probe set is used to detect TSC genes.
9. A kit for gene detection comprising the probe set of claim 8.
10. A gene sequencing method, comprising the steps of:
A) breaking the genome DNA in a sample to be detected into nucleic acid fragments and amplifying to construct a DNA library;
B) capturing in the DNA library a target sequence capable of specifically binding to the probe set using the probe set of claim 8;
C) sequencing the target sequence;
preferably, when N is 7P, the addition amount of the probe in the probe set is not less than 0.3 pmol.
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