CN116904583B - Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci - Google Patents

Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci Download PDF

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CN116904583B
CN116904583B CN202311159557.5A CN202311159557A CN116904583B CN 116904583 B CN116904583 B CN 116904583B CN 202311159557 A CN202311159557 A CN 202311159557A CN 116904583 B CN116904583 B CN 116904583B
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chr19
chr2
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CN116904583A (en
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李玉婷
张晓杰
李淑娟
高扬
陈迪
张秀娟
于倩
张丽
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Berry Genomics Co Ltd
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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
    • C12Q1/6876—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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Abstract

The invention discloses a detection probe set, a kit and a method for dynamic mutation of STR and VNTR gene loci, wherein the detection range comprises 73 gene loci, and the detection probe set relates to 79 diseases. The invention uses the targeted capturing long fragment and long reading long sequencing technology to realize the one-time detection of the target repeated area sequence information of all the targeted gene loci by a single sample, and overcomes the defects of single disease single detection, no target repeated area sequence information, small detectable range and long time required by differential diagnosis of the traditional detection technology.

Description

Detection probe set, kit and method for dynamic mutation of STR and VNTR gene loci
Technical Field
The invention relates to a detection probe set, detection equipment and a kit for dynamic mutation of STR and VNTR gene loci.
Background
Over 70 currently known dynamic mutant diseases are mainly related to nervous system diseases, and the total prevalence is about 1/1000. Along with the development of molecular genetic technology, the disease spectrum of the diseases is expanding continuously, but more than 70% of dynamic mutation diseases can not be diagnosed in time at present. In view of the wide variety and widespread high incidence of dynamic mutant diseases, the number of genes involved, new genes are continually being discovered, and the size and sequence conformation of pathogenic repeat amplifications are diverse, while our understanding of their underlying biological mechanisms remains largely inadequate, and thus there is an increasing need to improve molecular detection methods for aberrant repeat amplifications.
At present, common diagnostic methods for detecting dynamic mutation diseases include Southern blot, repeated primer PCR and second generation NGS sequencing. The Southern blot method is complex, time-consuming and labor-consuming, and has no sequence information of repeated regions of the pathogenic genes. The repeated primer PCR technology is simple and convenient, and even more than 1000 times of dynamic mutation diseases can indicate the existence of abnormal repeated amplification for the repeated sequence amplification times exceeding 100 times, but the exact length and sequence information cannot be obtained, and the specific primers/probes are required to be used for carrying out independent determination for each different gene.
The repeated region sequence information of the pathogenic genes is obtained to facilitate disease analysis. For example, spinocerebellar ataxia type 1 (SCA 1) is an autosomal dominant genetic disease, consisting ofATXN1Abnormal repeated amplification of CAG units in the gene. The presence of CAT interruption is important in diagnosing SCA1 patients with 39-44 repeated alleles, since only uninterrupted alleles are considered abnormal. Thus, in molecular diagnostics of such patients, it is desirable to analyze the number of repetitions of both CAG and CAT units simultaneously. The second generation NGS sequencing has a certain practicability in analyzing repeated amplification, and by using the STR signaling algorithm, it can be suggested whether the repetition number of part of genes exceeds the normal range, but the second scheme is still needed to verify. Many large fragment pathogenicity repeat amplifications, low complexity sequences, and high GC content fragments are difficult to analyze by NGS platforms, so second generation NGS sequencing does not explore the dynamic mutation-related variations well.
With the advent of third generation long-reading long-sequencing technology and the progress in computational analysis, the discovery speed of genes related to dynamic mutation diseases is remarkably improved. Only between 2019 and 2021, 17 new pathogenicity and risk-related tandem repeat amplifications were published. Three-generation sequencing provides a powerful tool for finding longer and more complex repetitive sequences.
2023 hoped that group companies released the third generation dynamic mutation detection chip "Repeat Hunter" andrelated patent CN116042610a is disclosed. According to the patent content, first, in terms of probe design, the area extending 3 kb at the upper and lower stream of STR site is designed, and the average interval between the sites covered by every two adjacent probes is 1000 bp. Such probe design methods may not be suitable for regions with high homologous sequences or abnormal GC content, thereby affecting the capturing efficiency of individual genes. Second, in terms of genomic DNA fragmentation, the DNA fragments after disruption are 4.5 to 5.5. 5.5 kb. Genomic DNA is randomly disrupted and flanking regions are required for gene targeting, the shorter the length of fragmented DNA, the less template is the target repeat region comprising both flanking regions. Third, in sequencing library length, a qualified library requires a major band length of 2 kb or more. It is desirable that the group company perform 2 PCR amplifications in the library construction procedure, which may result in a library major band of less than 5 kb due to the short fragment's greater amplification advantage. Currently, there are reports in the literature PLIN4The abnormal amplification repeated area of the gene can reach more than 4 kb, so that the length of the main band of the library is increased, and the detection of the oversized abnormal repeated amplification sample is facilitated.
Disclosure of Invention
In view of the urgent need of rapid, accurate and comprehensive molecular diagnosis for repeated amplification of dynamic mutation diseases, which can be met by the current third-generation long-reading long-sequencing technology, the defects of single disease single detection, no target repeated region sequence information, small detectable range and long time required by differential diagnosis of the traditional detection technology are overcome.
The inventors found that the human genome contained more than 100 ten thousand annotated tandem repeats. Because of its reproducibility and abundance in the human genome, the target repeat region may not be suitable for designing probes, and the presence of high GC or high AT regions in abnormally repeated amplified samples is difficult to detect, the probe design regions are flanking regions of STR and VNTR gene loci, i.e., target repeat regions are captured using specific sequences of the flanking regions.
The inventors have found that in the dynamic mutational disease spectrum, the pathogenic repeat units are diverse, and the number of repetitions of the same unit varies widely from individual to individual, some even up to thousands. Thus, the longer the random fragmentation length of genomic DNA, the longer the length of the PCR amplification product of the captured template, and the longer the length of the third generation sequencing library, the more advantageous is the analysis of the sequence information of the pathogenic repeat region, especially for the detection of oversized abnormal repeated amplified samples.
The inventors found that short fragments have amplification advantages when long fragment amplification is performed on the capture templates because the randomly fragmented DNA is of different length. The amount of PCR product is related to both primer concentration and cycle number, as will be apparent to those skilled in the art. The library construction process of the invention does not contain pre-capture PCR, so that experimental operation steps can be reduced, namely, purification and pre-capture amplification processes are omitted, and amplification preference of pre-capture PCR products can be avoided.
The present inventors found that the use of Y-type adaptors, and no pre-capture PCR, allows the omission of a blocking sequence for blocking adaptors during hybridization, and does not have a large impact on capture efficiency, coverage and coverage uniformity, thereby saving hybridization capture costs.
Thus, in a first aspect, the present invention provides a probe set for detecting dynamically mutated STR and VNTR gene sites, targeting the following sites:
1) STR gene loci (69):ATXN1、ATXN2、ATXN3、CACNA1A、ATXN7、ATXN8OS/ATXN8、 ATXN10、PPP2R2B、TBP、BEAN1/TK2、NOP56、DAB1、ATN1、FXN、GLS、RFC1、AR、DMPK、CNBP、LRP12、 GIPC1、NUTM2B-AS1/LOC642361、PABPN1、C9orf72、NIPA1、TAF1、DMD、VWA1、SPAST、CSTB、 SAMD12、STARD7、MARCHF6、YEATS2、TNRC6A、RAPGEF2、NOTCH2NLC、FMR1、AFF2、AFF3、ZNF713、 C11ORF80、DIP2B、CBL、HTT、JPH3、PRDM12、PRNP、SOX3、ARX、ZIC2、HOXD13、EIF4A3、RUNX2、 HOXA13、FOXL2、ZIC3、TBX1、XYLT1、COMP、TCF4、PHOX2B、FGF14、RILPL1、THAP11、POLG；
2) VNTR gene locus (4):CACNA1C、PLIN4、ABCA7、WDR7。
further, wherein the probe set satisfies the following requirements:
1) 2 kb areas of the 5 'flanking region and the 3' flanking region of the STR and VNTR gene loci are covered, and a main body adopts a 1X tiling covering mode to design probes; for the region where the probe is not designed properly, if the sequence specificity is poor, the probe is not designed; the probe density is increased according to the following principle when the probe coverage area is small:
If the bases covered by the probes in the 2 kb areas of the two flanking areas are 700-1000 bp, designing the probes in a 2X tiled covering mode; if the bases covered by the probes in the 2 kb areas of the two flanking areas are 500-700 bp, designing the probes in a 3X tiled coverage mode; if the base covered by the probe in each of the 2 kb regions of the flanking regions is <500 bp, the probe is designed in a 4 Xtiled coverage manner.
2) The probe length is 80-120 bp, and the GC content is preferably 40% -60%.
In a specific embodiment, when designing STR gene locus probes in a 4 x tiled coverage mode, if the target repetitive region of the gene locus can be designed with probes, preferentially designing probes in a 1 x tiled coverage mode; or the 5 'flanking region and the 3' flanking region are extended by 1: 1 kb region to design probes, namely, the probes are designed in a mode of 1X tiling coverage in the 2-3kb regions of the 5 'flanking region and the 3' flanking region.
In a specific embodiment, for a VNTR gene locus, whether or not probe density is increased, if the target repeat region of the gene locus can be probed, the probe is preferably designed in a 1 x tiled coverage manner; or the 5 'flanking region and the 3' flanking region are extended by 1.sup. 1 kb region to the upstream and downstream of each other to design the probe, i.e. the 2-3kb region of the 5 'flanking region and the 3' flanking region is covered by 1X tiling.
In a specific embodiment, wherein the set of probes comprises all or at least one of the following 2230 sequences:
1) The 1 x tiled probe design area includes the following loci:
STR geneSite(s):ATXN1、ATXN2、ATXN3、CACNA1A、ATXN7、ATXN8OS/ATXN8、ATXN10、 PPP2R2B、TBP、BEAN1/TK2、NOP56、DAB1、ATN1、FXN、GLS、RFC1、AR、DMPK、CNBP、LRP12、GIPC1、 PABPN1、C9orf72、NIPA1、TAF1、DMD、VWA1、CSTB、SAMD12、MARCHF6、YEATS2、TNRC6A、RAPGEF2、 NOTCH2NLC、FMR1、AFF2、AFF3、ZNF713、C11ORF80、DIP2B、CBL、HTT、JPH3、PRDM12、PRNP、SOX3、 ARX、ZIC2、HOXD13、EIF4A3、RUNX2、HOXA13、FOXL2、ZIC3、TBX1、XYLT1、COMP、TCF4、PHOX2B、 FGF14、RILPL1、THAP11、POLG；
VNTR gene locus:CACNA1C、PLIN4、ABCA7、WDR7；
1 Xtiling probe specific probe sequences (1867 total):
chr1:1433798-1433918;chr1:1433915-1434035;chr1:1434033-1434153;chr1:1434150-1434270;chr1:1434271-1434391;chr1:1434388-1434508;chr1:1434505-1434625;chr1:1434622-1434742;chr1:1434743-1434863;chr1:1434860-1434980;chr1:1434978-1435098;chr1:1435095-1435215;chr1:1435216-1435336;chr1:1435333-1435453;chr1:1435451-1435571;chr1:1435568-1435688;chr1:1435689-1435809;chr1:1435806-1435926;chr1:1435924-1436044;chr1:1436041-1436161;chr1:1436162-1436282;chr1:1436279-1436399;chr1:1436396-1436516;chr1:1436513-1436633;chr1:1436634-1436754;chr1:1436751-1436871;chr1:1436869-1436989;chr1:1436986-1437106;chr1:1437107-1437227;chr1:1437224-1437344;chr1:1437342-1437462;chr1:1437459-1437579;chr1:1437580-1437700;chr1:1437698-1437818;chr1:57365392-57365512;chr1:57365513-57365633;chr1:57365629-57365749;chr1:57365746-57365866;chr1:57365862-57365982;chr1:57365983-57366103;chr1:57366099-57366219;chr1:57366216-57366336;chr1:57366332-57366452;chr1:57366453-57366573;chr1:57366569-57366689;chr1:57366686-57366806;chr1:57366802-57366922;chr1:57367241-57367361;chr1:57367358-57367478;chr1:57367474-57367594;chr1:57367595-57367715;chr1:57367711-57367831;chr1:57367828-57367948;chr1:57367944-57368064;chr1:57368065-57368185;chr1:57368181-57368301;chr1:57368768-57368888;chr1:57368884-57369004;chr1:57369005-57369125;chr1:149390842-149390962;chr1:149391545-149391665;chr1:149391661-149391781;chr1:149391782-149391902;chr1:149391898-149392018;chr1:149392015-149392135;chr1:149392131-149392251;chr1:149392252-149392372;chr1:149392368-149392488;chr1:149392485-149392605;chr1:149392601-149392721;chr1:149392722-149392842;chr11:66742937-66743057;chr11:66743055-66743175;chr11:66743172-66743292;chr11:66743293-66743413;chr11:66743411-66743531;chr11:66743528-66743648;chr11:66743646-66743766;chr11:66743767-66743887;chr11:66744119-66744239;chr11:66744240-66744360;chr11:66744357-66744477;chr11:66744475-66744595;chr11:66744592-66744712;chr11:66744713-66744833;chr11:66744831-66744951;chr11:66744948-66745068;chr11:66745066-66745186;chr11:66745187-66745307;chr11:66745304-66745424;chr11:66745422-66745542;chr11:66745539-66745659;chr11:66745660-66745780;chr11:66746133-66746253;chr11:119204643-119204763;chr11:119204764-119204884;chr11:119204882-119205002;chr11:119205000-119205120;chr11:119205118-119205238;chr11:119205239-119205359;chr11:119205357-119205477;chr11:119205474-119205594;chr11:119205592-119205712;chr11:119205713-119205833;chr11:119205831-119205951;chr11:119205948-119206068;chr11:119206066-119206186;chr11:119206187-119206307;chr11:119206305-119206425;chr11:119206423-119206543;chr11:119206541-119206661;chr11:119206662-119206782;chr11:119206780-119206900;chr11:119206897-119207017;chr11:119207015-119207135;chr11:119207136-119207256;chr11:119207254-119207374;chr11:119207371-119207491;chr11:119207489-119207609;chr11:119207610-119207730;chr11:119207728-119207848;chr11:119207845-119207965;chr11:119207963-119208083;chr11:119208084-119208204;chr11:119208203-119208323;chr12:2253791-2253911;chr12:2253907-2254027;chr12:2254024-2254144;chr12:2254140-2254260;chr12:2254261-2254381;chr12:2254377-2254497;chr12:2254494-2254614;chr12:2254610-2254730;chr12:2254731-2254851;chr12:2254847-2254967;chr12:2254964-2255084;chr12:2255080-2255200;chr12:2255201-2255321;chr12:2255317-2255437;chr12:2255434-2255554;chr12:2255550-2255670;chr12:2255671-2255791;chr12:2256090-2256210;chr12:2256206-2256326;chr12:2256323-2256443;chr12:2256439-2256559;chr12:2256560-2256680;chr12:2256676-2256796;chr12:2256793-2256913;chr12:2256909-2257029;chr12:2257030-2257150;chr12:2257146-2257266;chr12:2257263-2257383;chr12:2257616-2257736;chr12:2257733-2257853;chr12:2257849-2257969;chr12:2257970-2258090;chr12:2255791-2255911;chr12:2255880-2256000;chr12:2255970-2256090;chr12:6934716-6934836;chr12:6935193-6935313;chr12:6935313-6935433;chr12:6935432-6935552;chr12:6935552-6935672;chr12:6935671-6935791;chr12:6935790-6935910;chr12:6935910-6936030;chr12:6936029-6936149;chr12:6936148-6936268;chr12:6936268-6936388;chr12:6936387-6936507;chr12:6936506-6936626;chr12:6936626-6936746;chr12:6936745-6936865;chr12:6936865-6936985;chr12:6936984-6937104;chr12:6937103-6937223;chr12:6937223-6937343;chr12:6937342-6937462;chr12:6937461-6937581;chr12:6937581-6937701;chr12:6937700-6937820;chr12:6937819-6937939;chr12:6937939-6938059;chr12:6938058-6938178;chr12:6938178-6938298;chr12:6938297-6938417;chr12:6938416-6938536;chr12:6938536-6938656;chr12:6938655-6938775;chr12:50505053-50505173;chr12:50505169-50505289;chr12:50505286-50505406;chr12:50505402-50505522;chr12:50505523-50505643;chr12:50505639-50505759;chr12:50505756-50505876;chr12:50505872-50505992;chr12:50505993-50506113;chr12:50506109-50506229;chr12:50506226-50506346;chr12:50506342-50506462;chr12:50506463-50506583;chr12:50506579-50506699;chr12:50506696-50506816;chr12:50506812-50506932;chr12:50506933-50507053;chr12:111596949-111597069;chr12:111597065-111597185;chr12:111597182-111597302;chr12:111597298-111597418;chr12:111597419-111597539;chr12:111597535-111597655;chr12:111597652-111597772;chr12:11159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X:148499304-148499424;chrX:148499420-148499540;chrX:148499541-148499661;chrX:148499657-148499777;chrX:148499774-148499894;chrX:148499890-148500010;chrX:148500011-148500131;chrX:148500127-148500247;chrX:148500244-148500364;chrX:148500360-148500480;chrX:148500481-148500601;chrX:148500743-148500863;chrX:148500859-148500979;chrX:148500976-148501096;chrX:148501092-148501212;chrX:148501213-148501333;chrX:148501329-148501449;chrX:148501446-148501566;chrX:148501562-148501682;chrX:148501683-148501803;chrX:148501799-148501919;chrX:148501916-148502036;chrX:148502032-148502152;chrX:148502153-148502273;chrX:148502269-148502389;chrX:148502386-148502506;chrX:148502502-148502622;chrX:148502623-148502743;chr13:102159577-102159697;chr13:102159695-102159815;chr13:102159812-102159932;chr13:102159930-102160050;chr13:102160051-102160171;chr13:102160169-102160289;chr13:102160286-102160406;chr13:102160404-102160524;chr13:102160525-102160645;chr13:102160643-102160763;chr13:102160760-102160880;chr13:102160878-102160998;chr13:102160999-102161119;chr13:102161117-102161237;chr13:102161234-102161354;chr13:102161352-102161472;chr13:102161473-102161593;chr13:102161708-102161828;chr13:102161826-102161946;chr13:102161947-102162067;chr13:102162065-102162185;chr13:102162182-102162302;chr13:102162300-102162420;chr13:102162421-102162541;chr13:102162539-102162659;chr13:102162656-102162776;chr13:102162774-102162894;chr13:102162895-102163015;chr13:102163013-102163133;chr13:102163130-102163250;chr13:102163248-102163368;chr13:102163369-102163489;chr13:102163488-102163608;chr13:102163606-102163726;chr15:89331580-89331700;chr15:89331698-89331818;chr15:89331817-89331937;chr15:89331935-89332055;chr15:89332056-89332176;chr15:89332175-89332295;chr15:89332295-89332415;chr15:89332413-89332533;chr15:89332532-89332652;chr15:89332650-89332770;chr15:89332771-89332891;chr15:89332889-89333009;chr15:89333008-89333128;chr15:89333126-89333246;chr15:89333247-89333367;chr15:89333366-89333486;chr15:89333485-89333605;chr15:89333605-89333725;chr15:89333723-89333843;chr15:89333842-89333962;chr15:89333960-89334080;chr15:89334081-89334201;chr15:89334199-89334319;chr15:89334318-89334438;chr15:89334436-89334556;chr15:89334557-89334677;chr15:89334676-89334796;chr15:89334795-89334915;chr15:89334915-89335035;chr15:89335033-89335153;chr15:89335152-89335272;chr15:89335270-89335390;chr15:89335391-89335511;chr15:89335510-89335630;chr16:67841223-67841343;chr16:67841343-67841463;chr16:67841463-67841583;chr16:67841584-67841704;chr16:67841704-67841824;chr16:67841824-67841944;chr16:67841944-67842064;chr16:67842065-67842185;chr16:67842185-67842305;chr16:67842305-67842425;chr16:67842426-67842546;chr16:67842546-67842666;chr16:67842666-67842786;chr16:67842786-67842906;chr16:67842907-67843027;chr16:67843027-67843147;chr16:67843147-67843267;chr16:67843267-67843387;chr16:67843388-67843508;chr16:67843508-67843628;chr16:67843628-67843748;chr16:67843749-67843869;chr16:67843869-67843989;chr16:67843989-67844109;chr16:67844109-67844229;chr16:67844230-67844350;chr16:67844350-67844470;chr16:67844470-67844590;chr16:67844590-67844710;chr16:67844711-67844831;chr12:123531838-123531958;chr12:123531956-123532076;chr12:123532074-123532194;chr12:123532195-123532315;chr12:123532313-123532433;chr12:123532430-123532550;chr12:123532548-123532668;chr12:123532669-123532789;chr12:123532787-123532907;chr12:123532905-123533025;chr12:123533023-123533143;chr12:123533144-123533264;chr12:123533262-123533382;chr12:123533379-123533499;chr12:123533497-123533617;chr12:123533618-123533738;chr12:123533736-123533856;chr12:123533854-123533974;chr12:123533972-123534092;chr12:123534093-123534213;chr12:123534211-123534331;chr12:123534328-123534448;chr12:123534446-123534566;chr12:123534567-123534687;chr12:123534685-123534805;chr19:4510446-4510566;chr19:4510561-4510681;chr19:4510675-4510795;chr19:4510790-4510910;chr19:4510911-4511031;chr19:4511026-4511146;chr19:4511140-4511260;chr19:4511255-4511375;chr19:4511376-4511496;chr19:4511491-4511611;chr19:4511605-4511725;chr19:4511720-4511840;chr19:4511841-4511961;chr19:4511956-4512076;chr19:4512070-4512190;chr19:4512185-4512305;chr19:4512306-4512426;chr19:4512420-4512540;chr19:4512535-4512655;chr19:4512649-4512769;chr19:4512770-4512890;chr19:4512885-4513005;chr19:4512999-4513119;chr19:4513114-4513234;chr19:4513235-4513355;chr19:4513352-4513472;chr19:4513468-4513588;chr19:4513584-4513704;chr7:55884919-55885039;chr7:55885040-55885160;chr7:55889640-55889760;chr7:55889746-55889866;chr7:55889853-55889973;chr7:55889959-55890079;chr7:55890520-55890640;
2) The 2 x tiled probe design area includes the following loci:
STR gene locus:TBP、FXN、NUTM2B-AS1/LOC642361、NIPA1、SPAST、CSTB、YEATS2、 NOTCH2NLC、ZNF713、PRDM12、EIF4A3；
the specific sequence of the 2 x tiling probe is as follows (217 in total):
chr1:149388722-149388842;chr1:149388782-149388902;chr1:149388842-149388962;chr1:149388902-149389022;chr1:149388962-149389082;chr1:149389022-149389142;chr1:149389082-149389202;chr1:149389142-149389262;chr1:149389202-149389322;chr1:149389262-149389382;chr1:149389322-149389442;chr1:149389382-149389502;chr1:149389442-149389562;chr1:149389502-149389622;chr1:149389562-149389682;chr1:149389622-149389742;chr1:149389682-149389802;chr1:149389742-149389862;chr1:149389802-149389922;chr1:149389862-149389982;chr1:149390762-149390882;chr10:79824234-79824354;chr10:79824294-79824414;chr10:79824354-79824474;chr10:79824414-79824534;chr10:79824474-79824594;chr10:79824534-79824654;chr10:79824594-79824714;chr10:79824654-79824774;chr10:79824714-79824834;chr10:79824774-79824894;chr10:79825014-79825134;chr10:79825074-79825194;chr10:79825134-79825254;chr10:79825194-79825314;chr10:79825254-79825374;chr10:79825314-79825434;chr10:79825374-79825494;chr10:79825434-79825554;chr10:79825494-79825614;chr10:79827344-79827464;chr10:79827404-79827524;chr10:79827464-79827584;chr10:79827524-79827644;chr10:79827584-79827704;chr10:79827644-79827764;chr10:79827704-79827824;chr10:79828064-79828184;chr10:79828124-79828244;chr10:79828184-79828304;chr10:79828244-79828364;chr10:79828304-79828424;chr10:79828364-79828484;chr15:22784771-22784891;chr15:22785491-22785611;chr15:22785551-22785671;chr15:22785611-22785731;chr15:22786091-22786211;chr15:22786151-22786271;chr15:22786211-22786331;chr15:22786271-22786391;chr15:22786331-22786451;chr15:22786391-22786511;chr15:22786451-22786571;chr15:22786511-22786631;chr15:22786571-22786691;chr15:22786631-22786751;chr17:80147039-80147159;chr17:80147099-80147219;chr17:80147159-80147279;chr17:80147219-80147339;chr17:80147279-80147399;chr17:80147339-80147459;chr17:80147399-80147519;chr17:80147459-80147579;chr17:80147519-80147639;chr17:80147579-80147699;chr17:80147639-80147759;chr17:80147699-80147819;chr17:80147999-80148119;chr17:80148059-80148179;chr17:80148119-80148239;chr17:80148359-80148479;chr17:80148419-80148539;chr2:32071717-32071837;chr2:32071777-32071897;chr2:32071837-32071957;chr2:32072317-32072437;chr2:32072377-32072497;chr2:32072557-32072677;chr2:32072617-32072737;chr2:32072677-32072797;chr2:32072737-32072857;chr2:32072797-32072917;chr2:32072857-32072977;chr2:32072917-32073037;chr2:32072977-32073097;chr2:32073037-32073157;chr2:32073097-32073217;chr2:32073277-32073397;chr2:32073337-32073457;chr2:32073397-32073517;chr2:32073577-32073697;chr2:32073637-32073757;chr2:32073697-32073817;chr2:32073757-32073877;chr2:32073817-32073937;chr2:32073877-32073997;chr2:32073937-32074057;chr2:32073997-32074117;chr2:32074057-32074177;chr2:32074117-32074237;chr2:32074177-32074297;chr2:32074237-32074357;chr2:32074297-32074417;chr2:32074357-32074477;chr2:32074417-32074537;chr2:32074777-32074897;chr2:32074837-32074957;chr2:32074897-32075017;chr2:32074957-32075077;chr2:32075017-32075137;chr21:43776399-43776519;chr21:43776459-43776579;chr21:43776519-43776639;chr21:43776579-43776699;chr21:43776639-43776759;chr21:43776699-43776819;chr21:43776759-43776879;chr21:43776819-43776939;chr21:43776879-43776999;chr21:43777959-43778079;chr21:43778019-43778139;chr21:43778079-43778199;chr21:43778139-43778259;chr21:43778199-43778319;chr21:43778259-43778379;chr21:43778319-43778439;chr21:43778379-43778499;chr21:43778439-43778559;chr3:183712446-183712566;chr3:183712506-183712626;chr3:183712566-183712686;chr3:183712626-183712746;chr3:183712686-183712806;chr3:183713046-183713166;chr3:183713106-183713226;chr3:183713166-183713286;chr3:183713526-183713646;chr3:183713586-183713706;chr3:183713646-183713766;chr3:183713706-183713826;chr6:170559826-170559946;chr6:170559886-170560006;chr6:170559946-170560066;chr6:170560006-170560126;chr6:170560966-170561086;chr6:170561266-170561386;chr6:170561326-170561446;chr6:170561386-170561506;chr6:170561446-170561566;chr6:170561506-170561626;chr6:170561566-170561686;chr6:170561626-170561746;chr6:170561686-170561806;chr6:170561746-170561866;chr6:170561806-170561926;chr7:55885581-55885701;chr7:55885641-55885761;chr7:55885701-55885821;chr7:55885761-55885881;chr7:55885821-55885941;chr7:55885881-55886001;chr7:55885941-55886061;chr7:55887081-55887201;chr7:55887141-55887261;chr7:55887201-55887321;chr7:55887261-55887381;chr7:55887321-55887441;chr7:55887381-55887501;chr7:55887441-55887561;chr7:55887501-55887621;chr9:69037474-69037594;chr9:69037534-69037654;chr9:69037594-69037714;chr9:69037894-69038014;chr9:69037954-69038074;chr9:69038014-69038134;chr9:69038074-69038194;chr9:69038134-69038254;chr9:69038194-69038314;chr9:69038254-69038374;chr9:69038314-69038434;chr9:69038374-69038494;chr9:69038434-69038554;chr9:69038734-69038854;chr9:69038794-69038914;chr9:69038854-69038974;chr9:69039214-69039334;chr9:69039274-69039394;chr9:130679945-130680065;chr9:130680665-130680785;chr9:130680725-130680845;chr9:130680785-130680905;chr9:130680845-130680965;chr9:130680905-130681025;chr9:130680965-130681085;chr9:130681025-130681145;chr9:130681085-130681205;chr9:130681145-130681265;chr9:130681205-130681325;chr9:130681265-130681385;chr9:130681325-130681445;chr9:130681385-130681505;chr9:130681445-130681565;chr9:130681505-130681625;chr9:130681565-130681685;
3) The 3 x tiled probe design area includes the following loci:
STR gene locus:ATXN3、STARD7、DIP2B；
VNTR gene locus:PLIN4；
the specific sequences of the 3 x tiling probes are (total 109):
chr12:50502921-50503041;chr12:50502961-50503081;chr12:50503001-50503121;chr12:50503041-50503161;chr12:50503081-50503201;chr12:50503761-50503881;chr12:50503801-50503921;chr12:50503841-50503961;chr12:50503881-50504001;chr12:50504521-50504641;chr12:50504561-50504681;chr12:50504601-50504721;chr12:50504641-50504761;chr12:50504681-50504801;chr12:50504721-50504841;chr12:50504761-50504881;chr12:50504801-50504921;chr12:50504841-50504961;chr12:50504881-50505001;chr12:50504921-50505041;chr12:50504961-50505081;chr14:92068930-92069050;chr14:92068970-92069090;chr14:92069010-92069130;chr14:92070050-92070170;chr14:92070090-92070210;chr14:92070130-92070250;chr14:92070170-92070290;chr14:92070210-92070330;chr14:92070570-92070690;chr14:92070610-92070730;chr14:92070730-92070850;chr14:92070770-92070890;chr14:92070810-92070930;chr14:92070850-92070970;chr14:92070890-92071010;chr14:92070930-92071050;chr14:92070970-92071090;chr19:4508527-4508647;chr19:4508567-4508687;chr19:4508607-4508727;chr19:4508647-4508767;chr19:4508687-4508807;chr19:4508727-4508847;chr19:4508767-4508887;chr19:4508807-4508927;chr19:4508847-4508967;chr19:4508887-4509007;chr19:4508927-4509047;chr19:4508967-4509087;chr19:4510327-4510447;chr19:4510367-4510487;chr19:4510407-4510527;chr19:4510447-4510567;chr19:4513501-4513621;chr19:4513541-4513661;chr19:4513581-4513701;chr19:4513621-4513741;chr19:4513661-4513781;chr19:4513701-4513821;chr19:4513741-4513861;chr19:4513781-4513901;chr19:4513821-4513941;chr19:4513861-4513981;chr19:4513901-4514021;chr19:4513941-4514061;chr19:4513981-4514101;chr19:4514021-4514141;chr19:4514061-4514181;chr19:4514101-4514221;chr19:4515541-4515661;chr2:96194975-96195095;chr2:96195015-96195135;chr2:96195055-96195175;chr2:96195095-96195215;chr2:96195135-96195255;chr2:96195175-96195295;chr2:96195215-96195335;chr2:96195255-96195375;chr2:96195295-96195415;chr2:96195335-96195455;chr2:96195375-96195495;chr2:96195415-96195535;chr2:96195455-96195575;chr2:96195495-96195615;chr2:96195535-96195655;chr2:96195575-96195695;chr2:96195615-96195735;chr2:96195655-96195775;chr2:96195695-96195815;chr2:96195735-96195855;chr2:96197415-96197535;chr2:96197455-96197575;chr2:96197495-96197615;chr2:96197535-96197655;chr2:96197575-96197695;chr2:96197935-96198055;chr2:96197975-96198095;chr2:96198375-96198495;chr2:96198415-96198535;chr2:96198455-96198575;chr2:96198495-96198615;chr2:96198775-96198895;chr2:96198815-96198935;chr2:96198855-96198975;chr2:96198895-96199015;chr2:96198935-96199055;chr2:96199055-96199175;chr2:96199095-96199215;
4) The 4 x tiled probe design area includes the following gene loci:
STR gene locus:ATXN10、ZNF713；
specific sequences of the 4 x tiling probes are (total 37):
chr22:45795329-45795449;chr22:45796769-45796889;chr22:45796799-45796919;chr22:45796829-45796949;chr22:45796859-45796979;chr22:45796889-45797009;chr22:45796919-45797039;chr22:45796949-45797069;chr22:45796979-45797099;chr22:45797009-45797129;chr22:45797039-45797159;chr22:45797069-45797189;chr22:45797099-45797219;chr22:45797129-45797249;chr22:45797159-45797279;chr22:45797189-45797309;chr22:45797219-45797339;chr7:55887874-55887994;chr7:55887904-55888024;chr7:55887934-55888054;chr7:55887964-55888084;chr7:55887994-55888114;chr7:55888024-55888144;chr7:55888054-55888174;chr7:55888084-55888204;chr7:55888114-55888234;chr7:55888204-55888324;chr7:55888234-55888354;chr7:55888264-55888384;chr7:55888294-55888414;chr7:55889434-55889554;chr7:55889464-55889584;chr7:55889494-55889614;chr7:55889524-55889644;chr7:55889554-55889674;chr7:55889584-55889704;chr7:55889614-55889734。
the probes of the invention are designed according to human reference genome version hg38, wherein each probe in the probe set is at least one of DNA double strand, DNA single strand and RNA single strand; the forward sequence and the reverse complementary sequence of the DNA double-stranded probe can be used for capturing dynamic mutation STR and VNTR gene loci.
Each probe in the probe set of the invention is labeled. The label is used for signal amplification and luminescence detection after specific binding of the probe to the fragmented DNA, and comprises at least one of biotin, avidin, an antibody or a chemical coupling agent, preferably biotin.
In a second aspect, the present invention provides a method of constructing a single molecule long read long sequencing library comprising the steps of:
1) Randomly fragmenting the DNA;
2) Performing terminal repair and pre-library linker ligation on the fragmented DNA to obtain a pre-library;
3) Hybridizing a pre-library with the probe set of the invention in the absence of a blocking sequence to obtain a hybridization product;
4) Capturing the hybridization product by using magnetic beads, cleaning, and performing PCR amplification to obtain a capturing library;
5) Three generation sequencing libraries were constructed.
In one embodiment, genomic DNA may be from a variety of sources, such as blood, blood spots, semen, sperm spots, bone, hair, saliva spots, sweat, tissue, and the like. The skilled person is aware of methods of disrupting genomic DNA, for example by sonication, mechanical disruption or by enzymatic cleavage, etc., and the disruption conditions may be adjusted according to the actual need.
In one embodiment, the fragmented DNA is 5-15 kb, preferably 7-10 kb in length.
In one embodiment, the pre-library linker is of Y-type structure, which may be a pendant T linker or a blunt end linker. "Y-type adaptor" refers to an adaptor formed by annealing two DNA single strands which are not completely complementary, one end of the adaptor forming a double strand due to base complementarity of the two strands, and the other end not forming a double strand due to incomplete complementarity between the bases of the two strands. For example, the sequences of the two chains comprised by the Y-type linker useful in the present invention are as follows:
F：
5’ -AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATC- 3’ (SEQ ID NO. 1)
R: (5' -terminal p represents a phosphorylation modification)
5’ -pGATCGGAAGAGCACACGTCTGAACTCCAGTCACATCTCGTATGCCGTCTTCTGCTTG- 3’ (SEQ ID NO. 2)
Wherein the underlined indicates the base complementary pairing of the two single strands.
In the context of the present invention, "blocking sequence" refers to a sequence for blocking a linker and/or tag sequence, including sequences designed to be reverse-complementary to the linker and/or tag sequence. In some embodiments, to increase the blocking effect of the blocking sequence, specific modifications are added at the end of the blocking sequence, such as inverted dT modifications, amino modifications, ddNTP modifications (including ddCTP, ddATP, ddGTP and ddTTP), spacer modifications, hypoxanthine modifications, random base modifications, and the like.
Based on the findings of the present inventors, better capture efficiency can be achieved without the need for pre-capture PCR pre-amplification and the use of Y-junctions, without the need for adding any blocking sequences to the hybridization system. Thus, in one embodiment, a pre-library is obtained without performing pre-capture PCR amplification. In another embodiment, the system for hybridization includes hybridization buffers, enhancement fluids, human Cot-1 DNA, and hybridization probes, but does not include a blocking sequence. The conditions for hybridization capture, such as hybridization temperature, hybridization time, heat wash temperature, and heat wash times, can be adjusted by those skilled in the art according to actual needs. The general principles of designing and preparing hybridization probes are also well known to those skilled in the art.
In one embodiment, the magnetic beads are used to capture hybridization products, preferably streptavidin magnetic beads.
In one embodiment, the post-capture PCR amplification system comprises an amplification enzyme, an amplification buffer, a PCR additive, amplification primers, and post-capture magnetic beads. The amplification enzyme has high fidelity and excellent amplification performance, and is suitable for amplifying complex templates such as long fragments, high GC/AT content and the like; the PCR additive can reduce the secondary structure, enhance the enzyme stability or inhibit non-specific amplification, and is selected from at least one of dimethyl sulfoxide, betaine, bovine serum albumin and formamide; the final concentration range of the amplification primer is 0.1-1 mu M, and the amplification primer is provided with phosphorylation modification and is adapted to a pre-library joint; the amplification procedure included: 94 ℃ for 2-4min;98℃for 10-15sec, 30sec at Tm for 30sec,68℃for 5-10min,15-25cycles; and the temperature is 68 ℃ for 5-10min.
In one embodiment, the major band of the post-capture PCR amplification products and the third generation sequencing library is about 8 kb.
In a specific embodiment, SMRT sequencing was performed using multiple dynamic mutation sequencing libraries, and single chip off-chip data indicated that the overall insert length was concentrated between 5 and 10 kb, as shown in FIG. 1.
In a third aspect, the present invention provides an apparatus for detecting the number of repetitions of STR and VNTR gene loci, comprising:
1) And a capturing module: for constructing a capture library;
2) Library construction module: the method is used for constructing a single-molecule long-reading long sequencing library;
3) Sequencing and analysis module: and carrying out third-generation sequencing on the sequencing library, and carrying out data analysis to obtain the sequence information of the target repetitive region.
In one embodiment, the set of probes used by the apparatus of the invention to construct the capture library is selected from the set of probes described above.
In one embodiment, the method used by the apparatus of the present invention to construct a capture library and a single molecule long read long sequencing library is selected from the sequencing library construction methods as described above.
In a specific embodiment, wherein the single molecule long read long sequencing platform is selected from the group consisting of a Pacific Biosciences company SMRT sequencing platform and an Oxford Nanopore company Nanopore single molecule sequencing platform based on single molecule real time sequencing technology.
In one embodiment, the data analysis flow of the apparatus of the present invention comprises:
1) After the third generation sequencing machine-down data is split by a sample, comparing the human genome reference sequences;
2) Reserving a unique comparison sequence, and screening a target area;
3) And counting the number of target repeated area sequences, capturing efficiency and coverage depth.
In one embodiment, the method for detecting the number of STR gene locus repetitions comprises:
1) Analyzing the repeated area according to the sequence of the target area, calculating the repeated times and the number of the supporting sequences, and judging pathogenicity according to the information recorded by the database;
2) And according to the specific sequence comparison of the 5 '-flanking region and the 3' -flanking region of the target repeated region, cutting the repeated region sequence, analyzing the repeated unit times and the typing clusters to obtain the repeated structure of each allele, and counting the repeated times of each allele and the sequence number supporting the allele.
In a specific embodiment, if the number of repeat times of both alleles of the STR gene locus detected by the above method is the same, the haplotypes are resolved based on SNVs phasing of the flanking regions of the repeat region; for two alleles with identical repeat structure, the number of repeats and the number of supporting sequences are counted separately.
In a specific embodiment, if the STR gene locus positive high repeat disruption detected by the above method is followed, the single-sided repeat sequence is flanked by repeat regions, either upstream or downstream of the repeat regions is located, sheared, and the number of repeat units is counted, supplementing the pathogenicity determination.
In a specific embodiment, the method for detecting the number of STR locus repeats, the output of the number of allele repeats can be selected from the following statistics: average, third quartile, median, mode, or peak returned by kernel density estimation, preferably median.
In one embodiment, the method for detecting the number of VNTR gene locus repetitions is:
1) Because of the large number of polymorphisms in the VNTR repeat unit, the detection of the VNTR locus repeat region requires construction of a repeat type list for each VNTR gene according to the existing sample set;
2) Cutting the sequence of the repeated region according to the specific sequence comparison of the 5 'flanking region and the 3' flanking region of the target repeated region, identifying repeated units from a built-in VNTR gene repeated type list, combining the same types of polymorphism and calculating the repeated times;
3) Similar to STR repeats, the typing clusters, counting the number of repeats and the number of supporting sequences per allele.
In a specific embodiment, the output of the number of allele repetitions in the method for detecting the number of VNTR gene locus repetitions may be selected from the following statistics: average, third quartile, median, mode, or peak returned by kernel density estimation, preferably median.
The method for detecting the repetition times of the dynamic mutation STR and VNTR gene loci can report the repetition times of the known pathogenic repeated units and can also report the repetition times of other repeated units in the repeated region. These other repeat units result from a comprehensive search of the database and published articles, which may suggest a relationship between the reported repeat units, but not yet recorded in the database or the pathogenic relationship is not yet clear, and the disease, which may help to fully explore repeat mutations that may lead to the disease.
In a fourth aspect, the invention provides a method for detecting the number of repetitions of dynamically mutated STR and VNTR gene loci, comprising the steps of:
1) Randomly fragmenting a DNA sample, repairing the tail ends and connecting the joints of the pre-library to obtain a pre-library;
2) Under the condition that no closed sequence exists, the probe set is hybridized with a pre-library, a hybridization product is captured by using magnetic beads, and PCR amplification is carried out after washing, so that a captured library is obtained;
3) And constructing a third-generation sequencing library, performing third-generation sequencing, and analyzing sequencing data to obtain target sequence information.
In one embodiment, the methods of the invention for constructing a pre-library, a capture library, and a third generation sequencing library are selected from the methods of constructing a single molecule long read long sequencing library as described above.
In one embodiment, the method of the invention for analyzing sequencing data is selected from the data analysis methods as described above.
In a fifth aspect, the invention provides a kit for detecting dynamically mutated STR and VNTR gene loci comprising the following components:
1) The probe set of the invention;
2) Reagents for constructing the pre-library;
3) Reagents for hybrid capture, excluding blocking sequences;
4) Reagents for PCR amplification;
5) Reagents for constructing third generation sequencing libraries.
In one embodiment, the reagents for constructing a pre-library include a repair enzyme, a repair buffer, a ligase, a ligation buffer, and a pre-library adaptor. In a specific embodiment, the pre-library linker is of Y-type structure, which may be a pendant T linker or a blunt end linker.
In one embodiment, the hybridization capture reagent includes hybridization buffer, enhancement solution, human Cot-1 DNA, the probe set of the present invention, magnetic beads, and a wash reagent, but does not include a blocking sequence.
In one embodiment, the magnetic beads are used to capture hybridization products, preferably streptavidin magnetic beads.
In one embodiment, the PCR amplification reagents include an amplification enzyme, an amplification buffer, a PCR additive, and an amplification primer. The amplification enzyme has high fidelity and excellent amplification performance, and is suitable for amplifying complex templates such as long fragments, high GC/AT content and the like; the PCR additive can reduce the secondary structure, enhance the enzyme stability or inhibit non-specific amplification, and is selected from at least one of dimethyl sulfoxide, betaine, bovine serum albumin and formamide; the final concentration range of the amplification primer is 0.1-1 mu M, and the amplification primer is provided with phosphorylation modification and is adapted to the pre-library joint.
In one embodiment, the reagents for constructing a third generation sequencing library include a repair enzyme, a ligase, an exonuclease, and a mating buffer, as well as a third generation sequencing linker.
In a sixth aspect, the present invention also provides the use of the probe set or the single-molecule long-read long-sequencing library construction method or the detection apparatus for the number of times of repetition of STR and VNTR gene loci or the detection method for the number of times of repetition of STR and VNTR gene loci in the preparation of a kit for detecting dynamic mutant STR and VNTR gene loci.
In one embodiment, the STR and VNTR gene locus kit is suitable for detecting one or more of the following gene loci:
1) STR gene loci (69):ATXN1、ATXN2、ATXN3、CACNA1A、ATXN7、ATXN8OS/ATXN8、 ATXN10、PPP2R2B、TBP、BEAN1/TK2、NOP56、DAB1、ATN1、FXN、GLS、RFC1、AR、DMPK、CNBP、LRP12、 GIPC1、NUTM2B-AS1/LOC642361、PABPN1、C9orf72、NIPA1、TAF1、DMD、VWA1、SPAST、CSTB、 SAMD12、STARD7、MARCHF6、YEATS2、TNRC6A、RAPGEF2、NOTCH2NLC、FMR1、AFF2、AFF3、ZNF713、 C11ORF80、DIP2B、CBL、HTT、JPH3、PRDM12、PRNP、SOX3、ARX、ZIC2、HOXD13、EIF4A3、RUNX2、 HOXA13、FOXL2、ZIC3、TBX1、XYLT1、COMP、TCF4、PHOX2B、FGF14、RILPL1、THAP11、POLG；
2) VNTR gene locus (4):CACNA1C、PLIN4、ABCA7、WDR7。
in one embodiment, the probe set or the detection device for the number of repetitions of the STR and VNTR gene sites or the detection kit for the dynamic mutation of STR and VNTR gene sites of the present invention can be used to detect one or more of the following diseases: spinocerebellar ataxia type 1 (SCA 1), spinocerebellar ataxia type 2 (SCA 2), spinocerebellar ataxia type 3 (SCA 3), spinocerebellar ataxia type 6 (SCA 6), spinocerebellar ataxia type 7 (SCA 7), spinocerebellar ataxia type 8 (SCA 8), spinocerebellar ataxia type 12 (SCA 12), spinocerebellar ataxia type 17 (SCA 17), spinocerebellar ataxia type 36 (SCA 36), dentate nuclear pallidoluffing (DRPLA), kennedy's disease (SBMA), myotonic muscular dystrophy type 1 (DM 1), huntington's Disease (HD), huntington-like disease-2 (HDL 2), hypopharyngeal muscular dystrophy (OPMD), spinocerebellar ataxia type 37 (SCA 37), friedrily ataxia (FRDA), systemic retardation, progressive ataxia, elevated glutamine (GDPAG), fragile X-related tremor/ataxia syndrome (FXTAS), fragile X Syndrome (FXS), fragile X-related ovarian dysfunction (FXPOI), fragile X-related psychotic disorder (FXAND), fragile XE syndrome (FRAXE), KINSSHIP syndrome (FRA 2A), dysnoesia FRA11 type (FRA 11A), dysnoesia FRA12 type (FRA 12A), jacobsen syndrome (FRA 11B), familial adult myoclonus type 1 (FAME 1), familial adult myoclonus type 6 (FAME 6), dysnoesia type 1 (FRA 2A), dysnoesia type 11A (FRA 11B), familial adult myoclonus type 7 (FAME 7), early infant epileptic encephalopathy type 1 (ELEE 1)/palington's mental retardation syndrome (PRTS)/x-related mental retardation with or without epileptic seizure (MRXARX), myotonic dystrophy type 2 (DM 2), neuronal nuclear inclusion body disease (NIID), hereditary essential tremor type 6 (ETM 6), oculopharyngeal myopathy type 3 (OPDM 3), oculopharyngeal myopathy type 1 (OPDM 1), oculopharyngeal myopathy type 2 (OPDM 2), oculopharyngeal disease with leukopathy (OPML 1), amyotrophic lateral sclerosis 1 (ALS 1), duchenne Muscular Dystrophy (DMD), hereditary Motor Neuropathy (HMN), amyotrophic lateral sclerosis 1 (ALS 1) hereditary sensory and autonomic neuropathy type 8 (HSAN 8), spastic paraplegia 4 (SPG 4), riceri-Costa-Pereira syndrome (RCPS), creutzfeldt-jakob disease (CJD), fuchs endothelial corneal dystrophy type 3 (FECD 3), multifinger malformation type 1, short finger and craniocaudal hypoplasia (CCD), hand-foot-reproductive syndrome (HFGS), palpebral fissure syndrome (BPES), full forebrain malformation type 5 (HPE 5), single growth hormone deficiency with mental developmental retardation (MRGH), x-linked hypophysis function (phph), congenital Central Hypoventilation Syndrome (CCHS), congenital joint malformation (VACTERL), eye-ear-spinal malformation (OVAS), farloquay (ToF), amenorrhea, epiphyseal dysplasia type 1 (EDM 1), pseudo-chondrohypoplasia (PSACH), oculopharyngeal myopathy type 4 (OPDM 4), late spinocerebellar ataxia-27B (SCA 27B), baratela-Scott syndrome (BSS), alzheimer's Disease (AD), schizophrenia and bipolar affective disorder (BD/SCZ), pansy positive autophagic rim-vacuolar Myopathy (MRUPAV), X-linked dystonia parkinson (XDP), amyotrophic lateral sclerosis 3 (ALS 3), spinocerebellar ataxia type 10 (SCA 10), spinocerebellar ataxia type 31 (SCA 31), cerebellar ataxia with neuropathy and disappearance of vestibular reflex (caas), autism spectrum disorders (FRA 7A), progressive myoclonus type 1 (EPM 1), familial myoclonus type 2 (fag 2), familial adult myoclonus type 3 (fastlujo), fascian focal fascian type 3 (fas-fastlujo), fascian type 4/fas-fastlujo (fas), fastluy focal fastluy type 4 (fas-fastluj), spine-fasdie (fastluj) and spine-4.
The invention discloses a probe set, detection equipment and a kit for detecting dynamic mutation STR and VNTR gene loci, which can detect target repetitive region sequence information of all target gene loci at one time by a single sample based on a targeted capturing long fragment and long reading long sequencing technology and overcome the defects of single disease single detection, no target repetitive region sequence information, small detectable range and long time required by differential diagnosis of the traditional detection technology.
Drawings
Fig. 1: sequencing library off-machine data.
Fig. 2: sequencing library construction flow chart.
Fig. 3: sequencing library fragment analysis, lower refers to Lower Marker.
Fig. 4: in sample 31PLIN4Target repeat region information of the gene.
Fig. 5: in sample 32PLIN4Target repeat region information of the gene.
Fig. 6: in sample 33PLIN4Target repeat region information of the gene.
Description of the embodiments
The invention described generally herein will be more readily understood by reference to the following examples, which one skilled in the art would be able to adapt by reference to the teachings herein to effect appropriate modification of the process parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be within the scope of the present invention. The following examples are provided by way of illustration and are not intended to limit the invention. These examples are not intended to be an indication that the experiments below are all or only experiments performed.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are all commercially available.
The invention provides a probe set, detection equipment and a kit for detecting dynamic mutation STR and VNTR gene loci, which realize that 73 STR and VNTR gene loci are detected at one time by a single sample through a targeted capturing long fragment and long reading long sequencing technology, and are beneficial to rapid, accurate and comprehensive molecular diagnosis in clinic, and the probe set, the detection equipment and the kit are used for detecting the dynamic mutation STR and VNTR gene loci. The correspondence between 73 dynamic mutation gene loci and the 79 diseases involved is shown in table 1.
Table 1: correspondence of 73 dynamic mutation gene loci to 79 diseases involved
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Example 1: three-generation sequencing library was constructed and the effect of different DNA initiation amounts on the main band of the library was tested
The three-generation sequencing library construction flow is shown in FIG. 2, and the specific construction method is as follows:
1. genomic DNA fragmentation
Genomic DNA fragmentation was performed using g-TUBE (Covaris, cat. No. 520079). And (3) using the same sample DNA, wherein the initial quantity is respectively 2 mug, 3 mug and 4 mug, and obtaining the fragmented DNA through AMPure PB magnetic bead purification after finishing the fragmentation reaction.
2. End repair
The reaction system shown in Table 2 was prepared using an end repair reagent (Enzymotics, cat. Y9140-HC-L) to complete the end repair reaction. The reaction procedure: 25. 30 minutes at the temperature; 75. 20 minutes at the temperature; and then maintained at 4 ℃.
Table 2: terminal repair system
3. Pre-library linker ligation
The ligation reaction was completed by preparing a reaction system shown in Table 3 using ligase reagent (enzymes, cat. L6030-HC-L). The reaction procedure: 20. 30 minutes at the temperature; and then maintained at 4 ℃. After the ligation reaction was completed, purification was performed using AMPure PB magnetic beads.
Table 3: connection system
4. Pre-library hybridization
Hybridization reagents (IDT, cat. No. 1072281) as shown in Table 4 were added to the purified product, and the mixture was thoroughly mixed and incubated at room temperature for 10 minutes. After incubation, the supernatant was transferred to a new low adsorption 0.2 mL centrifuge tube and 4 μl of the dynamic mutation probe set provided by the present invention was added. After thoroughly mixing, the mixture was centrifuged instantaneously and the following procedure was run on a PCR instrument: 95. 5 minutes at the temperature; 47 ℃ for 17 hours; and then maintained at 47 ℃.
Table 4: hybridization system
5. Magnetic bead capture and washing
The hybridization product was captured using streptavidin magnetic beads, and subjected to hot washing and room temperature washing (IDT, cat. No. 1072281), after which the magnetic beads were resuspended using enzyme-free water.
6. PCR amplification
To the above product, a reaction system (TOYOBO, cat. No. KFX-201) as shown in Table 5 was added, and after thoroughly mixing, the mixture was subjected to instantaneous centrifugation, and the procedure as shown in Table 6 was run. After the completion of the PCR reaction, purification was performed using AMPure PB beads.
Table 5: PCR reaction system
Table 6: PCR reaction procedure
7. Third generation sequencing library construction
A third generation sequencing library (PacBIO, cat# 102-088-900) was constructed using a kit, reagents including repair enzyme, ligase, exonuclease and supporting buffers, and a third generation sequencing linker, to finally obtain a sequencing library.
8. Library QC and results display
Fragment analysis of the library using the Agilent 4200 TapeStation system and genomic DNA reagents (Agilent, 5067-5365, 5067-5366) showed that 2-4 μg DNA starting amounts all made it possible to construct the library with about 8 kb for the main bands of the library, as shown in Table 7 and FIG. 3.
Table 7: library fragment analysis results
Example 2: constructing a third-generation sequencing library, and verifying STR gene locus detection performance
Referring to the library construction method of example 1, a sequencing library was constructed using 27 clinical samples and 3 Coriell human genome standard samples, respectively, and the number of repetitions of the different repeat units was counted as a median and compared with the RP-PCR or dmWES detection results or Coriell reference results. As shown in Table 8, the number of times of repetition detected by the method of the present invention was consistent with the result of RP-PCR or dmWES detection or the result of Coriell reference.
The method of the invention can report the repetition times of the known pathogenic repeated units and the repetition times of other repeated units in the repeated area. These other repeat units result from a comprehensive search of the database and published articles, which may suggest a relationship between the reported repeat units, but not yet recorded in the database or the pathogenic relationship is not yet clear, and the disease, which may help to fully explore repeat mutations that may lead to the disease.
Table 8: and (5) detecting STR gene locus. In sample 1 tested by the method of the present invention, "ATXN1CAG, CAT 27_1, 60_1 "represents autosomalATXN1One allele had 27 CAG units and 1 CAT unit, and the other allele had 60 CAG units and 1 CAT unit; the number of repetitions of the RP-PCR assay may include other interrupt sequences, such as "in sample 1"ATXN1CAG, CAT 28, 61 "means autosomalATXN1One allele had 28 CAG units (containing CAT units) and the other allele had 61 CAG units (containing CAT units); as below, "/" indicates undetected.
Note that: when the detection upper limit of RP-PCR or dmWES is not exceeded, the result is consistent within +/-2 of the repeated times deviation of the repeated units, and when the detection upper limit is exceeded, the theoretical pathogenicity judgment is consistent; samples 28 to 30 are respectively Coriell standard sample NA07537 # FMR1|CGG 28-29；>200）、NA07862（FMR1CGG 501-550) and NA09237FMR1CGG 931-940), the theoretical pathogenicity decisions are consistent, i.e., as a result.
Example 3: constructing a third-generation sequencing library, and verifying VNTR gene locus detection performance
Referring to the library construction method of example 1, sequencing libraries were constructed using clinical samples of 3 cases of known long fragment PCR results from hospitals, respectively, and VNTR gene loci were detectedPLIN4And comparing the target repetitive region information with the long fragment PCR amplification result. As shown in Table 9 and FIGS. 4-6, the length of the repeat region detected by the method of the present invention was consistent with the result of PCR amplification of the long fragment, and samples 32-33 all had abnormal repeated amplified bands.
Table 9:PLIN4gene locus detection results
Note that: the target repeat region length was considered to be nearly identical to the PCR product size.

Claims (45)

1. A probe set for detecting dynamically mutated STR and VNTR gene loci, targeting the following:
1) STR gene locus: ATXN1, ATXN2, ATXN3, CACNA1A, ATXN, ATXN8OS/ATXN8, ATXN10, PPP2R2B, TBP, BEAN1/TK2, NOP56, DAB1, ATN1, FXN, GLS, RFC1, AR, DMPK, CNBP, LRP, GIPC1, NUTM2B-AS1/LOC642361, PABPN1, C9ORF72, NIPA1, TAF1, DMD, VWA1, SPAST, CSTB, SAMD, STARD7, MARCHF6, YEATS2, TNRC6A, RAPGEF2, NOTCH2NLC, FMR1, AFF2, AFF3, ZNF, C11ORF80, DIP2B, CBL, HTT, JPH3, PRDM12, PRNP, SOX3, ARX, ZIC2, HOXD13, EIF4A3, RUNX2, HOXA13, xl2, ZIC3, TBX1, XYLT1, COMP 4, TCF 14, PHOX 1, phol 1, heat 11;
2) VNTR gene locus: CACNA1C, PLIN, ABCA7, WDR7;
wherein the probe set satisfies the following requirements:
1) Covering each 2kb region of the 5 'flanking region and the 3' flanking region of the STR and VNTR gene loci, and designing probes in a 1X tiled covering mode by a main body; for the region where the probe is not properly designed, the probe is not designed; the probe density is increased according to the following principle when the probe coverage area is small:
if the base covered by the probes in each 2kb region of the two flanking regions is 700-1000bp, designing the probes in a 2X tiled coverage mode; if the base covered by the probes in each 2kb region of the two flanking regions is 500-700bp, designing the probes in a 3X tiled coverage mode; if the base covered by the probes in the 2kb areas of the two flanking regions is less than 500bp, designing the probes in a 4X tiled coverage mode;
2) The probe length is 80-120bp, and the GC content is 40% -60%.
2. The probe set of claim 1, wherein when the probes are designed in a 4 x tiled manner for the STR gene locus, if the target repeat region of the gene locus can be designed, the probes are preferably designed in a1 x tiled manner; or the probe was designed in such a manner that the 2-3kb region of each of the 5 '-flanking region and the 3' -flanking region of the gene locus was covered with 1X tiling.
3. The set of probes according to claim 1, wherein for the VNTR gene locus, if the target repeat region of the gene locus can be probed, the probes are designed preferably in a1 x tiled coverage manner; or the probe was designed in such a manner that the 2-3kb region of each of the 5 '-flanking region and the 3' -flanking region of the gene locus was covered with 1X tiling.
4. A probe set according to any one of claims 1-3 comprising all or at least one of the following 2230 sequences:
1) The 1 x tiled probe design area includes the following loci:
STR gene locus: ATXN1, ATXN2, ATXN3, CACNA1A, ATXN, ATXN8OS/ATXN8, ATXN10, PPP2R2B, TBP, BEAN/TK 2, NOP56, DAB1, ATN1, FXN, GLS, RFC1, AR, DMPK, CNBP, LRP, GIPC1, PABPN1, C9ORF72, NIPA1, TAF1, DMD, VWA1, CSTB, SAMD12, MARCHF6, YEATS2, TNRC6A, RAPGEF2, NOTCH2NLC, FMR1, AFF2, AFF3, ZNF713, C11ORF80, DIP2B, CBL, HTT, JPH, PRDM12, PRNP, SOX3, ARX, ZIC2, HOXD13, EIF4A3, RUNX2, HOXA13, FOXL2, ZIC3, TBX1, XYLT1, COMP, TCF4, PHOX2B, FGF, lpl1, heat 11;
VNTR gene locus: CACNA1C, PLIN, ABCA7, WDR7;
1 Xtiling probe specific probe sequence:
chr1:1433798-1433918；chr1:1433915-1434035；chr1:1434033-1434153；chr1:1434150-1434270；chr1:1434271-1434391；chr1:1434388-1434508；chr1:1434505-1434625；chr1:1434622-1434742；chr1:1434743-1434863；chr1:1434860-1434980；chr1:1434978-1435098；chr1:1435095-1435215；chr1:1435216-1435336；chr1:1435333-1435453；chr1:1435451-1435571；chr1:1435568-1435688；chr1:1435689-1435809；chr1:1435806-1435926；chr1:1435924-1436044；chr1:1436041-1436161；chr1:1436162-1436282；chr1:1436279-1436399；c hr1:1436396-1436516；chr1:1436513-1436633；chr1:1436634-1436754；chr1:1436751-1436871；chr1:1436869-1436989；chr1:1436986-1437106；chr1:1437107-1437227；chr1:1437224-1437344；chr1:1437342-1437462；chr1:1437459-1437579；chr1:1437580-1437700；chr1:1437698-1437818；chr1:57365392-57365512；chr1:57365513-57365633；chr1:57365629-57365749；chr1:57365746-57365866；chr1:57365862-57365982；chr1:57365983-57366103；chr1:57366099-57366219；chr1:57366216-57366336；chr1:57366332-57366452；chr1:57366453-57366573；chr1:57366569-57366689；chr1:57366686-57366806；chr1:57366802-57366922；chr1:57367241-57367361；chr1:57367358-57367478；chr1:57367474-57367594；chr1:57367595-57367715；chr1:57367711-57367831；chr1:57367828-57367948；chr1:57367944-57368064；chr1:57368065-57368185；chr1:57368181-57368301；chr1:57368768-57368888；chr1:57368884-57369004；chr1:57369005-57369125；chr1:149390842-149390962；chr1:149391545-149391665；chr1:149391661-149391781；chr1:149391782-149391902；chr1:149391898-149392018；chr1:149392015-149392135；chr1:149392131-149392251；chr1:149392252-149392372；chr1:149392368-149392488；chr1:149392485-149392605；chr1:149392601-149392721；chr1:149392722-149392842；chr11:66742937-66743057；chr11:66743055-66743175；chr11:66743172-66743292；chr11:66743293-66743413；chr11:66743411-66743531；chr11:66743528-66743648；chr11:66743646-66743766；chr11:66743767-66743887；chr11:66744119-66744239；chr11:66744240-66744360；chr11:66744357-66744477；chr11:66744475-66744595；chr11:66744592-66744712；chr11:66744713-66744833；chr11:66744831-66744951；chr11:66744948-66745068；chr11:66745066-66745186；chr11:66745187-66745307；chr11:66745304-66745424；chr11:66745422-66745542；chr11:66745539-66745659；chr11:66745660-66745780；chr11:66746133-66746253；chr11:119204643-119204763；chr11:119204764-119204884；chr11:119204882-119205002；chr11:119205000-119205120；c hr11:119205118-119205238；chr11:119205239-119205359；chr11:119205357-119205477；chr11:119205474-119205594；chr11:119205592-119205712；chr11:119205713-119205833；chr11:119205831-119205951；chr11:119205948-119206068；chr11:119206066-119206186；chr11:119206187-119206307；chr11:119206305-119206425；chr11:119206423-119206543；chr11:119206541-119206661；chr11:119206662-119206782；chr11:119206780-119206900；chr11:119206897-119207017；chr11:119207015-119207135；chr11:119207136-119207256；chr11:119207254-119207374；chr11:119207371-119207491；chr11:119207489-119207609；chr11:119207610-119207730；chr11:119207728-119207848；chr11:119207845-119207965；chr11:119207963-119208083；chr11:119208084-119208204；chr11:119208203-119208323；chr12:2253791-2253911；c hr12:2253907-2254027；chr12:2254024-2254144；chr12:2254140-2254260；c hr12:2254261-2254381；chr12:2254377-2254497；chr12:2254494-2254614；c hr12:2254610-2254730；chr12:2254731-2254851；chr12:2254847-2254967；c hr12:2254964-2255084；chr12:2255080-2255200；chr12:2255201-2255321；c hr12:2255317-2255437；chr12:2255434-2255554；chr12:2255550-2255670；c hr12:2255671-2255791；chr12:2256090-2256210；chr12:2256206-2256326；c hr12:2256323-2256443；chr12:2256439-2256559；chr12:2256560-2256680；c hr12:2256676-2256796；chr12:2256793-2256913；chr12:2256909-2257029；c hr12:2257030-2257150；chr12:2257146-2257266；chr12:2257263-2257383；c hr12:2257616-2257736；chr12:2257733-2257853；chr12:2257849-2257969；c hr12:2257970-2258090；chr12:2255791-2255911；chr12:2255880-2256000；c hr12:2255970-2256090；chr12:6934716-6934836；chr12:6935193-6935313；c hr12:6935313-6935433；chr12:6935432-6935552；chr12:6935552-6935672；c hr12:6935671-6935791；chr12:6935790-6935910；chr12:6935910-6936030；c hr12:6936029-6936149；chr12:6936148-6936268；chr12:6936268-6936388；c hr12:6936387-6936507；chr12:6936506-6936626；chr12:6936626-6936746；c hr12:6936745-6936865；chr12:6936865-6936985；chr12:6936984-6937104；c hr12:6937103-6937223；chr12:6937223-6937343；chr12:6937342-6937462；c hr12:6937461-6937581；chr12:6937581-6937701；chr12:6937700-6937820；c hr12:6937819-6937939；chr12:6937939-6938059；chr12:6938058-6938178；c hr12:6938178-6938298；chr12:6938297-6938417；chr12:6938416-6938536；c hr12:6938536-6938656；chr12:6938655-6938775；chr12:50505053-50505173；chr12:50505169-50505289；chr12:50505286-50505406；chr12:50505402-50505522；chr12:50505523-50505643；chr12:50505639-50505759；chr12:50505756-50505876；chr12:50505872-50505992；chr12:50505993-50506113；chr12:50506109-50506229；chr12:50506226-50506346；chr12:50506342-50506462；chr12:50506463-50506583；chr12:50506579-50506699；chr12:50506696-50506816；chr12:50506812-50506932；chr12:50506933-50507053；chr12:111596949-111597069；chr12:111597065-111597185；chr12:111597182-111597302；chr12:111597298-111597418；chr12:111597419-111597539；chr12:111597535-111597655；chr12:111597652-111597772；chr12:111597768-111597888；ch 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X:148499774-148499894；chrX:148499890-148500010；chrX:148500011-148500131；chrX:148500127-148500247；chrX:148500244-148500364；chrX:148500360-148500480；chrX:148500481-148500601；chrX:148500743-148500863；chrX:148500859-148500979；chrX:148500976-148501096；chrX:148501092-148501212；chrX:148501213-148501333；chrX:148501329-148501449；c hrX:148501446-148501566；chrX:148501562-148501682；chrX:148501683-148501803；chrX:148501799-148501919；chrX:148501916-148502036；chrX:148502032-148502152；chrX:148502153-148502273；chrX:148502269-148502389；chrX:148502386-148502506；chrX:148502502-148502622；chrX:148502623-148502743；chr13:102159577-102159697；chr13:102159695-102159815；chr13:102159812-102159932；chr13:102159930-102160050；chr13:102160051-102160171；chr13:102160169-102160289；chr13:102160286-102160406；chr13:102160404-102160524；chr13:102160525-102160645；chr13:102160643-102160763；chr13:102160760-102160880；chr13:102160878-102160998；ch r13:102160999-102161119；chr13:102161117-102161237；chr13:102161234-102161354；chr13:102161352-102161472；chr13:102161473-102161593；chr13:102161708-102161828；chr13:102161826-102161946；chr13:102161947-102162067；chr13:102162065-102162185；chr13:102162182-102162302；chr13:102162300-102162420；chr13:102162421-102162541；chr13:102162539-102162659；chr13:102162656-102162776；chr13:102162774-102162894；chr13:102162895-102163015；chr13:102163013-102163133；chr13:102163130-102163250；chr13:102163248-102163368；chr13:102163369-102163489；chr13:102163488-102163608；chr13:102163606-102163726；chr15:89331580-89331700；chr15:89331698-89331818；chr15:89331817-89331937；chr15:89331935-89332055；chr15:89332056-89332176；chr15:89332175-89332295；chr15:89332295-89332415；chr15:89332413-89332533；chr15:89332532-89332652；chr15:89332650-89332770；chr15:89332771-89332891；chr15:89332889-89333009；chr15:89333008-89333128；chr15:89333126-89333246；chr15:89333247-89333367；chr15:89333366-89333486；chr15:89333485-89333605；chr15:89333605-89333725；chr15:89333723-89333843；chr15:89333842-89333962；chr15:89333960-89334080；chr15:89334081-89334201；chr15:89334199-89334319；chr15:89334318-89334438；chr15:89334436-89334556；chr15:89334557-89334677；chr15:89334676-89334796；chr15:89334795-89334915；chr15:89334915-89335035；chr15:89335033-89335153；chr15:89335152-89335272；chr15:89335270-89335390；chr15:89335391-89335511；chr15:89335510-89335630；chr16:67841223-67841343；chr16:67841343-67841463；chr16:67841463-67841583；chr16:67841584-67841704；chr16:67841704-67841824；chr16:67841824-67841944；chr16:67841944-67842064；chr16:67842065-67842185；chr16:67842185-67842305；chr16:67842305-67842425；chr16:67842426-67842546；chr16:67842546-67842666；chr16:67842666-67842786；chr16:67842786-67842906；chr16:67842907-67843027；chr16:67843027-67843147；chr16:67843147-67843267；chr16:67843267-67843387；chr16:67843388-67843508；chr16:67843508-67843628；chr16:67843628-67843748；chr16:67843749-67843869；chr16:67843869-67843989；chr16:67843989-67844109；chr16:67844109-67844229；chr16:67844230-67844350；chr16:67844350-67844470；chr16:67844470-67844590；chr16:67844590-67844710；chr16:67844711-67844831；chr12:123531838-123531958；chr12:123531956-123532076；chr12:123532074-123532194；chr12:123532195-123532315；chr12:123532313-123532433；chr12:123532430-123532550；chr12:123532548-123532668；chr12:123532669-123532789；chr12:123532787-123532907；chr12:123532905-123533025；chr12:123533023-123533143；chr12:123533144-123533264；chr12:123533262-123533382；chr12:123533379-123533499；chr12:123533497-123533617；chr12:123533618-123533738；chr12:123533736-123533856；chr12:123533854-123533974；chr12:123533972-123534092；chr12:123534093-123534213；chr12:123534211-123534331；chr12:123534328-123534448；chr12:123534446-123534566；chr12:123534567-123534687；chr12:123534685-123534805；chr19:4510446-4510566；chr19:4510561-4510681；chr19:4510675-4510795；chr19:4510790-4510910；chr19:4510911-4511031；chr19:4511026-4511146；chr19:4511140-4511260；chr19:4511255-4511375；chr19:4511376-4511496；chr19:4511491-4511611；chr19:4511605-4511725；chr19:4511720-4511840；chr19:4511841-4511961；chr19:4511956-4512076；chr19:4512070-4512190；chr19:4512185-4512305；chr19:4512306-4512426；chr19:4512420-4512540；chr19:4512535-4512655；chr19:4512649-4512769；chr19:4512770-4512890；chr19:4512885-4513005；chr19:4512999-4513119；chr19:4513114-4513234；chr19:4513235-4513355；chr19:4513352-4513472；chr19:4513468-4513588；chr19:4513584-4513704；chr7:55884919-55885039；chr7:55885040-55885160；chr7:55889640-55889760；chr7:55889746-55889866；chr7:55889853-55889973；chr7:55889959-55890079；chr7:55890520-55890640；
2) The 2 x tiled probe design area includes the following loci:
STR gene locus: TBP, FXN, NUTM2B-AS1/LOC642361, NIPA1, SPAST, CSTB, YEATS2, NOTCH2NLC, ZNF713, PRDM12, EIF4A3;
the specific sequence of the 2 x tiled probe is as follows:
chr1:149388722-149388842；chr1:149388782-149388902；chr1:149388842-149388962；chr1:149388902-149389022；chr1:149388962-149389082；chr1:149389022-149389142；chr1:149389082-149389202；chr1:149389142-149389262；chr1:149389202-149389322；chr1:149389262-149389382；chr1:149389322-149389442；chr1:149389382-149389502；chr1:149389442-149389562；chr1:149389502-149389622；chr1:149389562-149389682；chr1:149389622-149389742；chr1:149389682-149389802；chr1:149389742-149389862；chr1:149389802-149389922；chr1:149389862-149389982；chr1:149390762-149390882；chr10:79824234-79824354；chr10:79824294-79824414；chr10:79824354-79824474；chr10:79824414-79824534；chr10:79824474-79824594；chr10:79824534-79824654；chr10:79824594-79824714；chr10:79824654-79824774；chr10:79824714-79824834；chr10:79824774-79824894；chr10:79825014-79825134；chr10:79825074-79825194；chr10:79825134-79825254；chr10:79825194-79825314；chr10:79825254-79825374；chr10:79825314-79825434；chr10:79825374-79825494；chr10:79825434-79825554；chr10:79825494-79825614；chr10:79827344-79827464；chr10:79827404-79827524；chr10:79827464-79827584；chr10:79827524-79827644；chr10:79827584-79827704；chr10:79827644-79827764；chr10:79827704-79827824；chr10:79828064-79828184；chr10:79828124-79828244；chr10:79828184-79828304；chr10:79828244-79828364；chr10:79828304-79828424；chr10:79828364-79828484；chr15:22784771-22784891；chr15:22785491-22785611；chr15:22785551-22785671；chr15:22785611-22785731；chr15:22786091-22786211；chr15:22786151-22786271；chr15:22786211-22786331；chr15:22786271-22786391；chr15:22786331-22786451；chr15:22786391-22786511；chr15:22786451-22786571；chr15:22786511-22786631；chr15:22786571-22786691；chr15:22786631-22786751；chr17:80147039-80147159；chr17:80147099-80147219；chr17:80147159-80147279；chr17:80147219-80147339；chr17:80147279-80147399；chr17:80147339-80147459；chr17:80147399-80147519；chr17:80147459-80147579；chr17:80147519-80147639；chr17:80147579-80147699；chr17:80147639-80147759；chr17:80147699-80147819；chr17:80147999-80148119；chr17:80148059-80148179；chr17:80148119-80148239；chr17:80148359-80148479；chr17:80148419-80148539；chr2:32071717-32071837；chr2:32071777-32071897；chr2:32071837-32071957；ch r2:32072317-32072437；chr2:32072377-32072497；chr2:32072557-32072677；chr2:32072617-32072737；chr2:32072677-32072797；chr2:32072737-32072857；chr2:32072797-32072917；chr2:32072857-32072977；chr2:32072917-32073037；chr2:32072977-32073097；chr2:32073037-32073157；chr2:32073097-32073217；chr2:32073277-32073397；chr2:32073337-32073457；chr2:32073397-32073517；chr2:32073577-32073697；chr2:32073637-32073757；chr2:32073697-32073817；chr2:32073757-32073877；chr2:32073817-32073937；chr2:32073877-32073997；chr2:32073937-32074057；chr2:32073997-32074117；ch r2:32074057-32074177；chr2:32074117-32074237；chr2:32074177-32074297；chr2:32074237-32074357；chr2:32074297-32074417；chr2:32074357-32074477；chr2:32074417-32074537；chr2:32074777-32074897；chr2:32074837-32074957；chr2:32074897-32075017；chr2:32074957-32075077；chr2:32075017-32075137；chr21:43776399-43776519；chr21:43776459-43776579；chr21:43776519-43776639；chr21:43776579-43776699；chr21:43776639-43776759；chr21:43776699-43776819；chr21:43776759-43776879；chr21:43776819-43776939；chr21:43776879-43776999；chr21:43777959-43778079；chr21:43778019-43778139；chr21:43778079-43778199；chr21:43778139-43778259；chr21:43778199-43778319；chr21:43778259-43778379；chr21:43778319-43778439；chr21:43778379-43778499；chr21:43778439-43778559；chr3:183712446-183712566；chr3:183712506-183712626；chr3:183712566-183712686；chr3:183712626-183712746；chr3:183712686-183712806；chr3:183713046-183713166；chr3:183713106-183713226；chr3:183713166-183713286；chr3:183713526-183713646；chr3:183713586-183713706；chr3:183713646-183713766；chr3:183713706-183713826；chr6:170559826-170559946；chr6:170559886-170560006；c hr6:170559946-170560066；chr6:170560006-170560126；chr6:170560966-170561086；chr6:170561266-170561386；chr6:170561326-170561446；chr6:170561386-170561506；chr6:170561446-170561566；chr6:170561506-170561626；chr6:170561566-170561686；chr6:170561626-170561746；chr6:170561686-170561806；chr6:170561746-170561866；chr6:170561806-170561926；chr7:55885581-55885701；chr7:55885641-55885761；chr7:55885701-55885821；ch r7:55885761-55885881；chr7:55885821-55885941；chr7:55885881-55886001；chr7:55885941-55886061；chr7:55887081-55887201；chr7:55887141-55887261；chr7:55887201-55887321；chr7:55887261-55887381；chr7:55887321-55887441；chr7:55887381-55887501；chr7:55887441-55887561；chr7:55887501-55887621；chr9:69037474-69037594；chr9:69037534-69037654；chr9:69037594-69037714；chr9:69037894-69038014；chr9:69037954-69038074；chr9:69038014-69038134；chr9:69038074-69038194；chr9:69038134-69038254；chr9:69038194-69038314；chr9:69038254-69038374；chr9:69038314-69038434；ch r9:69038374-69038494；chr9:69038434-69038554；chr9:69038734-69038854；chr9:69038794-69038914；chr9:69038854-69038974；chr9:69039214-69039334；chr9:69039274-69039394；chr9:130679945-130680065；chr9:130680665-130680785；chr9:130680725-130680845；chr9:130680785-130680905；chr9:130680845-130680965；chr9:130680905-130681025；chr9:130680965-130681085；chr9:130681025-130681145；chr9:130681085-130681205；chr9:130681145-130681265；chr9:130681205-130681325；chr9:130681265-130681385；chr9:130681325-130681445；chr9:130681385-130681505；chr9:130681445-130681565；chr9:130681505-130681625；chr9:130681565-130681685；
3) The 3 x tiled probe design area includes the following loci:
STR gene locus: ATXN3, start 7, DIP2B;
VNTR gene locus: PLIN4;
the specific sequence of the 3 x tiling probe is as follows:
chr12:50502921-50503041；chr12:50502961-50503081；chr12:50503001-50503121；chr12:50503041-50503161；chr12:50503081-50503201；chr12:50503761-50503881；chr12:50503801-50503921；chr12:50503841-50503961；ch r12:50503881-50504001；chr12:50504521-50504641；chr12:50504561-50504681；chr12:50504601-50504721；chr12:50504641-50504761；chr12:50504681-50504801；chr12:50504721-50504841；chr12:50504761-50504881；chr12:50504801-50504921；chr12:50504841-50504961；chr12:50504881-50505001；ch r12:50504921-50505041；chr12:50504961-50505081；chr14:92068930-92069050；chr14:92068970-92069090；chr14:92069010-92069130；chr14:92070050-92070170；chr14:92070090-92070210；chr14:92070130-92070250；chr14:92070170-92070290；chr14:92070210-92070330；chr14:92070570-92070690；ch r14:92070610-92070730；chr14:92070730-92070850；chr14:92070770-92070890；chr14:92070810-92070930；chr14:92070850-92070970；chr14:92070890-92071010；chr14:92070930-92071050；chr14:92070970-92071090；chr19:4508527-4508647；chr19:4508567-4508687；chr19:4508607-4508727；chr19:4508647-4508767；chr19:4508687-4508807；chr19:4508727-4508847；chr19:4508767-4508887；chr19:4508807-4508927；chr19:4508847-4508967；chr19:4508887-4509007；chr19:4508927-4509047；chr19:4508967-4509087；chr19:4510327-4510447；chr19:4510367-4510487；chr19:4510407-4510527；chr19:4510447-4510567；chr19:4513501-4513621；chr19:4513541-4513661；chr19:4513581-4513701；chr19:4513621-4513741；chr19:4513661-4513781；chr19:4513701-4513821；chr19:4513741-4513861；chr19:4513781-4513901；chr19:4513821-4513941；chr19:4513861-4513981；chr19:4513901-4514021；chr19:4513941-4514061；chr19:4513981-4514101；chr19:4514021-4514141；chr19:4514061-4514181；chr19:4514101-4514221；chr19:4515541-4515661；chr2:96194975-96195095；chr2:96195015-96195135；chr2:96195055-96195175；chr2:96195095-96195215；chr2:96195135-96195255；chr2:96195175-96195295；ch r2:96195215-96195335；chr2:96195255-96195375；chr2:96195295-96195415；chr2:96195335-96195455；chr2:96195375-96195495；chr2:96195415-96195535；chr2:96195455-96195575；chr2:96195495-96195615；chr2:96195535-96195655；chr2:96195575-96195695；chr2:96195615-96195735；chr2:96195655-96195775；chr2:96195695-96195815；chr2:96195735-96195855；chr2:96197415-96197535；chr2:96197455-96197575；chr2:96197495-96197615；chr2:96197535-96197655；chr2:96197575-96197695；chr2:96197935-96198055；chr2:96197975-96198095；chr2:96198375-96198495；chr2:96198415-96198535；ch r2:96198455-96198575；chr2:96198495-96198615；chr2:96198775-96198895；chr2:96198815-96198935；chr2:96198855-96198975；chr2:96198895-96199015；chr2:96198935-96199055；chr2:96199055-96199175；chr2:96199095-96199215；
4) The 4 x tiled probe design area includes the following gene loci:
STR gene locus: ATXN10, ZNF713;
the specific sequence of the 4 x tiling probe is:
chr22:45795329-45795449；chr22:45796769-45796889；chr22:45796799-45796919；chr22:45796829-45796949；chr22:45796859-45796979；chr22:45796889-45797009；chr22:45796919-45797039；chr22:45796949-45797069；ch r22:45796979-45797099；chr22:45797009-45797129；chr22:45797039-45797159；chr22:45797069-45797189；chr22:45797099-45797219；chr22:45797129-45797249；chr22:45797159-45797279；chr22:45797189-45797309；chr22:45797219-45797339；chr7:55887874-55887994；chr7:55887904-55888024；chr7:55887934-55888054；chr7:55887964-55888084；chr7:55887994-55888114；ch r7:55888024-55888144；chr7:55888054-55888174；chr7:55888084-55888204；chr7:55888114-55888234；chr7:55888204-55888324；chr7:55888234-55888354；chr7:55888264-55888384；chr7:55888294-55888414；chr7:55889434-55889554；chr7:55889464-55889584；chr7:55889494-55889614；chr7:55889524-55889644；chr7:55889554-55889674；chr7:55889584-55889704；chr7:55889614-55889734。
5. the set of probes of any one of claims 1-4, wherein each probe in the set of probes is at least one of DNA double-stranded, DNA single-stranded, or RNA single-stranded; wherein both the forward sequence and the reverse complement of the DNA double-stranded probe can be used to capture the dynamic mutant STR and VNTR gene sites.
6. The set of probes according to any one of claims 1-5, wherein each probe in the set of probes is labeled.
7. The set of probes of claim 6, wherein the label is selected from one or more of the following group: biotin, avidin, antibodies or chemical coupling agents.
8. The set of probes of claim 6, wherein the label is biotin.
9. A method of constructing a single molecule long read long sequencing library comprising the steps of:
1) Randomly fragmenting the DNA sample;
2) Performing terminal repair and pre-library linker ligation on the fragmented DNA to obtain a pre-library;
3) Hybridizing the pre-library with a set of probes in the absence of blocking sequences to obtain hybridization products, wherein the set of probes is the set of probes of any one of claims 1-8;
4) Capturing the hybridization product by using magnetic beads, cleaning, and performing PCR amplification to obtain a capturing library;
5) Three generation sequencing libraries were constructed.
10. The method of claim 9, wherein the random fragmentation of DNA in step 1) comprises sonication, mechanical disruption, or by enzymatic cleavage.
11. The method of claim 9, wherein the randomly fragmented DNA in step 1) ranges from 5-15kb in length.
12. The method of claim 9, wherein the randomly fragmented DNA in step 1) ranges from 7-10kb in length.
13. The method of claim 9, wherein the pre-library linker in step 2) is of Y-type structure, which may be a pendant T linker or a blunt end linker.
14. The method of claim 13, wherein the pre-library adaptor comprises two DNA single strands which are not fully complementary, the length of the DNA single strands is 30-70bp, and the working adaptor is prepared after annealing reaction.
15. The method of claim 9, wherein the pre-library is obtained in step 2) without performing pre-capture PCR amplification.
16. The method of claim 9, wherein the hybridization system of step 3) further comprises hybridization buffer, enhancement solution, human Cot-1 DNA, but does not include a blocking sequence.
17. The method of claim 9, wherein the blocking sequence in step 3) refers to a sequence designed to be reverse-complementary to the linker sequence.
18. The method of claim 9, wherein the amplification system in step 4) comprises an amplification enzyme, an amplification buffer, PCR additives, amplification primers, and post-bead capture products.
19. The method of claim 18, wherein the amplification enzyme has high fidelity and excellent amplification properties suitable for amplifying long fragments and complex templates with high GC/AT content.
20. The method of claim 18, wherein the PCR additive can reduce secondary structure, enhance enzyme stability, or inhibit non-specific amplification; the PCR additive is at least one selected from dimethyl sulfoxide, betaine, bovine serum albumin and formamide.
21. The method of claim 18, wherein the amplification primer is in the final concentration range of 0.1-1 μm with a phosphorylation modification, fitting a pre-library adaptor; the amplification procedure included: 94 ℃ for 2-4min;98℃for 10-15sec, 30sec at Tm for 30sec,68℃for 5-10min,15-25cycles; and the temperature is 68 ℃ for 5-10min.
22. The method of claim 9, wherein the major band of the third generation sequencing library in step 5) is 5-10kb.
23. A device for detecting the number of STR and VNTR gene locus repetitions, comprising:
1) And a capturing module: a set of probes for constructing a capture library, wherein the set of probes for constructing a capture library is the set of probes of any one of claims 1-8;
2) Library construction module: the method is used for constructing a single-molecule long-reading long sequencing library;
3) Sequencing and analysis module: and carrying out third-generation sequencing on the sequencing library, and carrying out data analysis to obtain the sequence information of the target repetitive region.
24. The apparatus of claim 23, wherein the method of constructing a capture library and a single molecule long read long sequencing library is the method of any one of claims 9-22.
25. The apparatus of claim 23, wherein the single molecule long read long sequencing platform is selected from the group consisting of an SMRT sequencing platform and a nanopore single molecule sequencing platform based on single molecule real time sequencing technology.
26. The apparatus of claim 23, wherein the data analysis flow comprises:
1) After the third generation sequencing machine-down data is split by a sample, comparing the human genome reference sequences;
2) Reserving a unique comparison sequence, and screening a target area;
3) And counting the number of target repeated area sequences, capturing efficiency and coverage depth.
27. The process of claim 26 wherein the number of STR locus repetitions is detected by:
1) Analyzing the repeated area according to the sequence of the target area, calculating the repeated times and the number of the supporting sequences, and judging pathogenicity according to the information recorded by the database;
2) And according to the specific sequence comparison of the 5 '-flanking region and the 3' -flanking region of the target repeated region, cutting the repeated region sequence, analyzing the repeated unit times and the typing clusters to obtain the repeated structure of each allele, and counting the repeated times of each allele and the sequence number supporting the allele.
28. The method of claim 27, wherein the method of detecting the number of repetitions of the STR locus further comprises, when the number of repetitions of both alleles of the STR locus is the same, splitting haplotypes according to SNVs phasing of both flanking regions of the repeated region; for two alleles with identical repeat structure, the number of repeats and the number of supporting sequences are counted separately.
29. The method of any one of claims 27 or 28, wherein the method of detecting the number of repeats of an STR locus further comprises, when STR locus positive high repeats are interrupted, flanking the repeat region for a single repeat sequence, locating the repeat region upstream or downstream, shearing the filter, counting the number of repeat units, supplementing the pathogenicity determination.
30. The process of claim 26, wherein the number of VNTR gene locus repetitions is detected by:
1) Constructing a repeat type list for each VNTR gene according to the existing sample set;
2) Cutting the sequence of the repeated region according to the specific sequence comparison of the 5 'flanking region and the 3' flanking region of the target repeated region, identifying repeated units from a built-in VNTR gene repeated type list, combining the same types of polymorphism and calculating the repeated times;
3) Typing and clustering, and counting the repetition times and the number of supporting sequences of each allele.
31. A method for detecting the number of repetitions of dynamically mutated STR and VNTR loci comprising the steps of:
1) Randomly fragmenting a DNA sample, repairing the tail ends and connecting the joints of the pre-library to obtain a pre-library;
2) Hybridizing the probe set according to any one of claims 1-8 with a pre-library in the absence of a blocking sequence, capturing the hybridized products using magnetic beads, washing, and performing PCR amplification to obtain a captured library;
3) And constructing a third-generation sequencing library, performing third-generation sequencing, and analyzing sequencing data to obtain target sequence information.
32. The method of claim 31, wherein the methods of constructing a pre-library, capturing a library, and third generation sequencing library are the methods of any one of claims 9-22.
33. The method of claim 31, wherein the sequencing data analysis method is the method of any one of claims 26-30.
34. A kit for detecting dynamically mutated STR and VNTR gene loci comprising:
1) The set of probes of any one of claims 1-8;
2) Reagents for constructing the pre-library;
3) Reagents for hybrid capture, excluding blocking sequences;
4) Reagents for PCR amplification;
5) Reagents for constructing third generation sequencing libraries.
35. The kit of claim 34, wherein the reagents for constructing a pre-library comprise a repair enzyme, a repair buffer, a ligase, a ligation buffer, and a pre-library adaptor.
36. The kit of claim 35, wherein the pre-library linker is of Y-type structure, which may be a pendant T linker or a blunt end linker.
37. The kit of claim 34, wherein the hybridization capture reagent comprises hybridization buffer, enhancement solution, human Cot-1 DNA, the probe set of any one of claims 1-8, magnetic beads, and a wash reagent, but does not comprise a blocking sequence.
38. The kit of claim 37, wherein the blocking sequence refers to a sequence designed to be reverse-complementary to a linker sequence.
39. The kit of claim 34, wherein the PCR amplification reagents comprise an amplification enzyme, an amplification buffer, a PCR additive, and an amplification primer.
40. The kit of claim 39, wherein the amplification enzyme has high fidelity and excellent amplification properties, and is suitable for amplifying complex templates such as long fragments and high GC/AT content.
41. The kit of claim 39, wherein the PCR additive is selected from at least one of dimethyl sulfoxide, betaine, bovine serum albumin, and formamide, and can reduce secondary structure, enhance enzyme stability, or inhibit non-specific amplification.
42. The kit of claim 39, wherein the final concentration of the amplification primers is in the range of 0.1-1. Mu.M, with phosphorylation modifications, adapted to the pre-library adaptor.
43. The kit of claim 34, wherein the reagents for constructing a tertiary sequencing library comprise a repair enzyme, a ligase, an exonuclease and a companion buffer, and a tertiary sequencing linker.
44. Use of a set of probes according to any one of claims 1 to 8 or a method of single molecule long read long sequencing library construction according to any one of claims 9 to 22 or an apparatus according to any one of claims 23 to 30 or a method according to any one of claims 31 to 33 in the preparation of a kit for the detection of dynamic mutated STR and VNTR gene loci.
45. The use of claim 44, wherein said STR and VNTR gene loci comprise:
1) STR gene locus: ATXN1, ATXN2, ATXN3, CACNA1A, ATXN, ATXN8OS/ATXN8, ATXN10, PPP2R2B, TBP, BEAN1/TK2, NOP56, DAB1, ATN1, FXN, GLS, RFC1, AR, DMPK, CNBP, LRP, GIPC1, NUTM2B-AS1/LOC642361, PABPN1, C9ORF72, NIPA1, TAF1, DMD, VWA1, SPAST, CSTB, SAMD, STARD7, MARCHF6, YEATS2, TNRC6A, RAPGEF2, NOTCH2NLC, FMR1, AFF2, AFF3, ZNF, C11ORF80, DIP2B, CBL, HTT, JPH3, PRDM12, PRNP, SOX3, ARX, ZIC2, HOXD13, EIF4A3, RUNX2, HOXA13, xl2, ZIC3, TBX1, XYLT1, COMP 4, TCF 14, PHOX 1, phol 1, heat 11;
2) VNTR gene locus: CACNA1C, PLIN, ABCA7, WDR7.
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