CN111808855B - Construction method of universal gene detection library for hereditary familial hypercholesterolemia and kit thereof - Google Patents
Construction method of universal gene detection library for hereditary familial hypercholesterolemia and kit thereof Download PDFInfo
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Abstract
The invention relates to a construction method and a kit of a universal gene detection library aiming at hereditary familial hypercholesterolemia. The construction method and the kit are based on a universal high-throughput sequencing joint, relate to LDLR, APOB, PCSK9 and LDLRAP1 gene mutation, can be compatible with various sequencing platforms including Ion Torrent and Illumina platforms and the like, and have the advantages of simple and rapid library construction steps, cost saving, wide application range and the like.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a construction method and a kit of a universal gene detection library for hereditary familial hypercholesterolemia.
Background
Hereditary Familial Hypercholesterolemia (FH) is a disorder of lipid metabolism whose clinical and genetic characteristics were first defined, mainly manifested by a significant increase in serum low-density lipoprotein cholesterol (LDL-C) levels, and cutaneous/tendonoyellow tumors. The clinical classification is two subtypes of homozygous familial hypercholesterolemia (HoFH) and heterozygous familial hypercholesterolemia (HeFH). The homozygous type is rare, the morbidity is one millionth, the low-density lipoprotein cholesterol (LDL-C) value of a patient is usually 500-1200 mg/dL which is 4-6 times of that of a normal person, the patient has multiple myotendinoxanthoma and early atherosclerosis, and the severe person can have coronary heart disease in the adolescent period. The incidence of heterozygotes is 1/500, and although superficial features are not apparent, the rate of early atherosclerosis is not underestimated. Some patients develop aortic stenosis or dilatation-like and calcification of the heart valves, which can lead to valvular stenosis or insufficiency, ultimately leading to heart failure.
Hereditary familial hypercholesterolemia is a monogenic hereditary disease, mainly manifested by autosomal dominant inheritance. The mutations on the main pathogenic genes LDLR, APOB and PCSK9 can explain about 80 percent of FH patients. The Chinese specialist consensus on familial hypercholesterolemia screening and diagnosis and treatment indicates that the detection of pathogenic mutations in the LDLR, APOB, PCSK9 and LDLRAP1 genes as genetic diseases is the gold standard for FH diagnosis. Current data show that FH is known to be very poorly and diagnosed, resulting in poorer treatment. The probability of coronary heart disease in the population with FH but no treatment is about 20 times that of the ordinary population, so early screening and early drug treatment are critical to improving the survival rate of FH patients. The gene detection can help to diagnose early and differentiate the disease, guide clinical treatment to a certain extent, help to differentiate other high-risk relatives with disease risk in the family, guide good birth and good care and reduce the risk of disease again of offspring by analyzing whether the disease-related gene is mutated or not.
The hereditary familial hypercholesterolemia pathogenic gene is clear, the currently known pathogenic genes are LDLR, APOB, PCSK9 and LDLRAP1, but the pathogenic site is distributed in the whole exon region of the genes and has no hotspot mutation, so a relatively high-throughput inspection method is needed for detection. Currently, NGS sequencing methods are widely used for genetic disease detection with clear pathogenic genes but without hotspot mutations. The NGS sequencing usually adopts Massively Parallel Sequencing (MPS), can realize simultaneous sequencing of multiple samples and multiple sites, and greatly improves sequencing flux. High throughput sequencing technology has been demonstrated to have high accuracy and sensitivity in clinical gene testing. Currently, the NGS sequencing platforms widely applied in clinic are mainly Ion Torrent and Illumina, but due to different sequencing technical principles, different platforms adopt different technical processes during library construction, so that the libraries are not universal, namely, the libraries suitable for the Ion Torrent platform can not generate sequencing data on the Illumina platform, and vice versa. This has generated a great limitation to clinical application, so it is necessary to find a generic gene detection library for hereditary familial hypercholesterolemia, which is suitable for different sequencing platforms and meets different clinical requirements.
The invention is provided in view of the above.
Disclosure of Invention
In order to solve the technical problems, the invention mainly aims to provide a universal gene detection library preparation kit for hereditary familial hypercholesterolemia, which has simple, convenient and quick library construction steps, effectively reduces the cost and is suitable for different sequencing platforms.
Another object of the present invention is to provide a method for constructing the universal gene testing library for hereditary familial hypercholesterolemia.
Another object of the present invention is to provide a universal gene testing library for hereditary familial hypercholesterolemia obtained by the method.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention firstly provides a universal high-throughput sequencing linker for hereditary familial hypercholesterolemia, wherein the linker has the following sequence;
sequence a:
5’- XXXXXXTAGCTGAGTCGGAGACACGCAGGATCGGAAGAGCACGTCTGAACTC CAGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’；
sequence b:
5’- ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTGCGTGTCTC CGACTCAGCTAXXXXXX-3’；
sequence c:
5’- ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTCTCTATGGG CAGTCGGTGATXXXXXX-3’
sequence d:
5’- XXXXXXATCACCGACTGCCCATAGAGAGGATCGGAAGAGCACGTCTGAACTCC AGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’
where XXXXXXX represents the sample tag sequence.
In some embodiments, the tag sequence is selected from the group consisting of part or all of SEQ ID NO. 1-10.
In some embodiments, the linker sequence is annealed according to the following procedure and reaction system, annealing sequences a and b, respectively, to form a double-stranded linker, and annealing sequences c and d, respectively, to form another double-stranded linker.
The invention provides a kit for constructing a universal gene detection library for hereditary familial hypercholesterolemia, which comprises any one of the universal high-throughput sequencing adapters.
In some embodiments, the kit further comprises a PCR amplification primer set directed to the full coding region and/or variable splicing region of some or all of the genes LDLR, APOB, PCSK9, and LDLRAP 1.
In some preferred embodiments, the primer sequence is selected from the group consisting of part or all of the primer sequences in FIGS. 5-8.
In other preferred embodiments, the primer sets are further divided into two groups: the primer sequences for group 1 are shown in FIGS. 5-6, and the primer sequences for group 2 are shown in FIGS. 7-8.
The invention also provides an application of the universal high-throughput sequencing joint and the kit for the hereditary familial hypercholesterolemia in library construction or gene sequencing; preferably, the universal high-throughput sequencing adaptor for hereditary familial hypercholesterolemia is applied to library construction or gene sequencing.
The invention also provides a construction method of the universal gene detection library for hereditary familial hypercholesterolemia, which comprises the following steps:
1) multiplex PCR amplification: designing a synthesized FH primer pool, and performing multiplex PCR amplification on a target region;
2) and (3) digesting a primer sequence: carrying out digestion reaction on the primer amplification product obtained in the step 1);
3) universal linker preparation, preparing a universal high throughput sequencing linker according to any one of claims 1-2;
4) connecting a sequencing joint: connecting the products of the step 2) and the step 3) under the action of DNA ligase;
5) library purification and amplification: and (3) adding purified magnetic beads into the ligation product obtained in the step 4) for purification, and carrying out PCR amplification on the purified product to obtain a universal gene detection library for hereditary familial hypercholesterolemia.
In some embodiments, said step 1) designs a pool of synthetic primers against the full coding region and/or variable splicing region of part or all of the genes LDLR, APOB, PCSK9 and LDLRAP 1.
In some preferred embodiments, the primer sequence is selected from the group consisting of part or all of the primer sequences in FIGS. 5-8.
In other preferred embodiments, the primer sets are further divided into two groups: the primer sequences for group 1 are shown in FIGS. 5-6, and the primer sequences for group 2 are shown in FIGS. 7-8.
In some embodiments, the step 2) mixes the amplification products of the two independent primer pools obtained in the step 1), and adds the digestion reaction premix to the mixed PCR product in a volume ratio of 1: 10; because the two ends of the amplification product are primer sequences, the digestion reaction in the step 2) can avoid introducing degenerate basic groups at the 5' end of the primers in the amplification process, and reduce the sequencing length (the length of the primer sequences at the two ends is nearly 40 bp).
The invention also provides a universal gene detection library for hereditary familial hypercholesterolemia, which is prepared by the needle construction method.
Compared with the prior art, the invention has the following beneficial technical effects:
1. the universal sequencing adaptors of the invention are compatible with a variety of sequencing platforms, including paired double-stranded complementary regions and unpaired single-stranded free arms. The distal end of the paired double-stranded portion contains a tag sequence, and the non-free ends of the two free arms contain tag sequences. The base composition of the label sequences carried by the same sample is consistent, and whether cross contamination exists in the library building process can be judged according to the consistency of the label sequences. After sequencing by using an Illumina sequencing platform, analysis of sequencing data can judge whether index hopping occurs or not according to whether base compositions of tag sequences of the same reads are consistent or not. The distal end of the paired double-stranded part contains a tag sequence, and the bases of the tag sequences carried by the sense strand and the antisense strand of the same read are identical. A particular mutation is determined to be true by the presence of both the sense and antisense strands of the same read. If a certain read only has mutation in the sense strand or the antisense strand, the read can be judged as an error in the library construction or sequencing process, and the mutation cannot be included for subsequent analysis procedures, so that false positive is avoided. In addition, the universal sequencing joint designed by the invention can effectively overcome the inherent label jumping problem of a sequencing part platform, and can realize authenticity interpretation of low-frequency mutation and the like.
2. The universal gene detection library for hereditary familial hypercholesterolemia prepared by the method or the kit can be carried out on all types of sequencing platforms of Ion Torrent and Illumina to generate sequencing data. So that the library construction kit and the method are not limited by the existing sequencing platform. Meeting increasingly diverse clinical requirements. Meanwhile, the development cost and the development period of an application enterprise are saved.
3. The library provided by the invention adopts a high-throughput gene sequencing technology to detect gene mutation of LDLR, APOB, PCSK9 and LDLRAP1, the detection range is the whole coding region and the variable shearing region of the genes, the main disease genes of hereditary familial hypercholesterolemia are covered, and the mutation detection is comprehensive.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1, schematic structural diagram of a universal high throughput sequencing adapter described in example 1;
FIG. 2, bioanalyzer measurements (a-e) of 10 blood DNA libraries 2100 constructed according to the method of the present invention;
FIG. 3, bioanalyzer test results (f-j) of 10 blood DNA libraries 2100 constructed according to the method of the present invention;
FIG. 4, a library construction and assay flow diagram according to the present invention;
FIGS. 5-6, primer sequences of PCR multiplex amplification primer pool 1;
FIGS. 7-8, primer sequences of PCR multiplex amplification primer pool 2.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The following terms or definitions are provided only to aid in understanding the present invention. These definitions should not be construed to have a scope less than understood by those skilled in the art.
Unless defined otherwise below, all technical and scientific terms used in the detailed description of the present invention are intended to have the same meaning as commonly understood by one of ordinary skill in the art. While the following terms are believed to be well understood by those skilled in the art, the following definitions are set forth to better explain the present invention.
As used herein, the terms "comprising," "including," "having," "containing," or "involving" are inclusive or open-ended and do not exclude additional unrecited elements or method steps. The term "consisting of …" is considered to be a preferred embodiment of the term "comprising". If in the following a certain group is defined to comprise at least a certain number of embodiments, this should also be understood as disclosing a group which preferably only consists of these embodiments.
Where an indefinite or definite article is used when referring to a singular noun e.g. "a" or "an", "the", this includes a plural of that noun.
The terms "about" and "substantially" in the present invention denote an interval of accuracy that can be understood by a person skilled in the art, which still guarantees the technical effect of the feature in question. The term generally denotes a deviation of ± 10%, preferably ± 5%, from the indicated value.
Furthermore, the terms first, second, third, (a), (b), (c), and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.
The term "nucleic acid" or "nucleic acid sequence" in the present invention refers to any molecule, preferably polymeric molecule, comprising units of ribonucleic acid, deoxyribonucleic acid, or analogues thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a nucleic acid that denatures one strand of a double-stranded DNA. Alternatively, the single-stranded nucleic acid may be a single-stranded nucleic acid not derived from any double-stranded DNA.
The term "complementary" as used herein relates to hydrogen bonding base pairing between nucleotide bases G, A, T, C and U, such that when two given polynucleotides or polynucleotide sequences anneal to each other, a pairs with T, G pairs with C in DNA, G pairs with C, and a pairs with U in RNA.
Example 1 design and preparation of Universal high throughput sequencing adapters
Based on the prior patent application CN202010407833.5 of the applicant, a universal sequencing joint sequence which can be used for LDLR, APOB, PCSK9, LDLRAP1 and other genes and is simultaneously suitable for Ion Torrent and Illumina multi-sequencing platforms is designed and prepared. The specific method comprises the following steps:
based on the construction of the linker assembly shown in fig. 1, 10 sets of universal high throughput sequencing linkers AN1/PN1-AN10/PN10 were designed for the full coding region and the variable splicing region of the LDLR, APOB, PCSK9 and LDLRAP1 genes, with:
first synthesizing the following single chains
Sequence a:
5’- XXXXXXTAGCTGAGTCGGAGACACGCAGGATCGGAAGAGCACGTCTGAACTC CAGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’；
sequence b:
5’- ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTGCGTGTCTC CGACTCAGCTAXXXXXX-3’；
sequence c:
5’- ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTCTCTATGGG CAGTCGGTGATXXXXXX-3’
sequence d:
5’- XXXXXXATCACCGACTGCCCATAGAGAGGATCGGAAGAGCACGTCTGAACTCC AGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’
wherein XXXXXXX represents sample tag sequences, and specific groups 1-10 tag sequences are respectively:
annealing is carried out according to the following procedures and reaction systems, wherein the sequences a and b are respectively annealed to form AN 1-AN10 linkers, and the sequences c and d are respectively annealed to form PN 1-PN 10 linkers.
| Name of reagent | Volume of μ L |
| NEBuffer 10x | 1.25 |
| Single chain (50uM) | 5 |
| Single chain (50uM) | 5 |
| Nuclease-free water | 1.25 |
| Total | 12.5 |
And (3) annealing procedure: and (3) heating the sample to 105 ℃, annealing the sample by using a PCR instrument, denaturing the sample at 95 ℃ for 5min, then cooling the sample at 95 ℃ to 25 ℃, and adjusting the cooling speed to be tem/min which is 0.6%, thereby obtaining the universal high-throughput sequencing joint. In addition, if the stability of the linker is further enhanced, it is possible to select a stability modification, such as a thio modification, at the 3 'end of the free arm of the linker, preferably a phosphorothioate instead of the phosphodiester bond between the last 3 bases of the 3' end, and the like. Such as:
sequence a:
5’- XXXXXXTAGCTGAGTCGGAGACACGCAGGATCGGAAGAGCACGTCTGAACTC CAGTCACXXXXXXATCTCGTATGCCGTCTTC*T*G-3’；
sequence d:
5’- XXXXXXATCACCGACTGCCCATAGAGAGGATCGGAAGAGCACGTCTGAACTCC AGTCACXXXXXXATCTCGTATGCCGTCTTC*T*G-3’
example 2 Universal Gene testing library construction for hereditary familial hypercholesterolemia
10 samples of hereditary familial hypercholesterolemia patients are collected for library construction, a primer pool containing the full coding region and the variable splicing region (exon-intron extension is 20bp) of LDLR, APOB, PCSK9 and LDLRAP1 genes as target regions is used for carrying out multiple reaction PCR, and a sequencing joint is connected to construct a library, wherein the specific operation flow is as follows:
(1) nucleic acid extraction and quality inspection: after nucleic acid extraction and quality inspection, blood cells are required to meet certain quality control standards: the DNA concentration is 10 ng/. mu.L; DNA purity: OD 260/2801.8-2.0, OD260/230> 2; total initial amount of DNA was 20 ng.
(2) Multiplex PCR primer design and amplification:
a primer pool based on the whole coding region and the variable splicing region (exon-intron extension 20bp) of LDLR, APOB, PCSK9 and LDLRAP1 genes is designed, and a primer combination for the genes is established through a large number of screening optimization experiments in the early stage of the invention. In addition, to ensure that the target region (the entire coding region of the gene to be tested, as well as the variable splicing region) is completely covered and to avoid primer dimers or short fragments forming between the primers of adjacent amplicons, the primers are separated into two separate primer pools: the specific sequences of primer pools 1 and 2 can be seen in FIGS. 5-8.
The PCR amplification primers were separated into two separate primer pools for multiplex PCR amplification with 10. mu.L of each reaction and a total volume of 20. mu.L. Performing multiple PCR amplification on the DNA meeting the DNA quality standard in the step (1), wherein the reaction system is as follows: the amplification reaction premix 2. mu.L, FH primer pool 5. mu.L, DNA 3. mu.L, total 10. mu.L. The multiplex PCR reaction conditions were: the reaction was terminated by holding at 99 ℃ for 2min and then performing 16 cycles of 99 ℃ for 15s, 60 ℃ for 4min and then 10 ℃.
(3) And (3) digesting a primer sequence: mixing the amplification products of the two independent primer pools obtained in the step (2), and adding the amplification products into a digestion reaction system as follows: mu.L of amplification product + 2. mu.L of digestion reaction premix, total volume 22. mu.L. Digestion reaction conditions are as follows: maintaining at 50 deg.C for 20min,55 deg.C for 20min, 60 deg.C for 20min, and then at 10 deg.C for no more than 1 h.
(4) Connecting a universal joint: reagents were added to the digest in the following order: 4. mu.L of ligation buffer, 2. mu.L of ligase, 1. mu.L of AN linker and 1. mu.L of PN linker prepared in example 1, and 8. mu.L in total; reaction conditions are as follows: maintaining at 22 deg.C for 30 min; keeping at 68 deg.C for 5 min; keeping at 72 deg.C for 5 min; finally keeping the temperature at 10 ℃ for not more than 1 h.
(5) Purifying the ligation product obtained in the step (4) by using a purified magnetic bead, and then carrying out PCR amplification according to the following procedures and systems:
and purifying the amplified library to obtain the universal gene detection library for hereditary familial hypercholesterolemia.
Example 3 Multi-platform sequencing verification and detection
Respectively combined with high-throughput sequencing platform Ion GeneStaudioTMDNA sequencing is carried out on an S5 Plus platform and a Miseq DX platform, and then point mutation (SNP) and small fragment insertion deletion (InDel) are detected, and the specific steps are as follows:
(1) purification the library was then quality checked and quantified using Agilent 2100 and QUBIT 4.0. The 2100 quality inspection map of the library is shown in a figure 2-3, and shows that the main peak of the library length fragment is near 400bp and the main peak of the library is a single sharp single peak, and the result shows that the two ends of the target gene fragment are connected with the universal high-throughput sequencing adaptor. And calculating to obtain the concentration of the library according to the dilution multiple, wherein the concentration of the library is higher than 1ng/uL, so that subsequent experimental steps can be carried out, and the library construction fails below 1 ng/uL.
(2) Sequencing and verifying the universal gene detection library of the hereditary familial hypercholesterolemia by respectively adopting an Ion Torrent platform and an Illumina platform from the library obtained in the step (1), and specifically comprising the following steps:
A. ion Torrent platform Ion GeneStaudioTMSequencing on an S5 Plus sequencer, and specifically implementing the following steps:
purifying and inspecting the aboveLibrary dilution with Ion 520TM&Ion 530TMKit-OT, according to the Kit operating protocol, after template preparation on an IonTouch 2 instrument, Ion GeneStaudioTMSequencing and data analysis were performed on an S5 Plus gene sequencer.
B. The method comprises the following specific steps of carrying out on-machine sequencing on an Illumina platform Miseq DX sequencer:
the above purification and quality control was diluted with a library, performed according to the Kit protocol using Miseq DX Reagent Kit v3, and subjected to sequencing and data analysis on a Miseq DX gene sequencer.
Analysis of sequencing data results was performed as follows:
for Ion GeneStaudioTMS5 Plus platform:
1) analysis of Ion GeneStaudioTMThe off-line result of the S5 Plus platform mainly comprises the base number more than or equal to Q20, the average read length of the read, On Target, Uniformity and Mean depth. In particular, see the following table:
2) the average length of the read segments of the samples is more than or equal to 200bp, which indicates that all the samples of the samples are read through, namely, the bases between the head and the tail of the target fragment to be detected can be identified; mean depth is more than or equal to 450 times, which indicates that the target fragments to be detected are sequenced more than 450 times; the On targets are all more than or equal to 98 percent, which indicates that 98 percent of the measured base sequences can be compared in the range of the Target area to be measured, and the Uniformity is all more than or equal to 93 percent, which indicates that the amplification efficiency of each read in the Target area to be measured and the efficiency of connecting a universal high-throughput joint are similar. The parameters indicate that the two ends of the target gene segment to be detected of the sample are successfully connected with the universal high-throughput sequencing joint, and the sequencing is successful; the universal gene detection library for indicating hereditary familial hypercholesterolemia can be used in Ion GeneStaudioTMSequencing was performed by the S5 Plus gene sequencer.
For the Miseq DX platform:
1) the Miseq DX platform offline results were analyzed and mainly included data yield, Reads counts, and Q30 percentages.
As shown in the table below.
2) The sample data yield is more than or equal to 0.4G, the Reads data is more than or equal to 3M, and the Q30 percentage is more than or equal to 83%, which indicates that the sample sequencing is successful; the two ends of the target gene segment to be detected of the sample are successfully connected with the universal high-throughput sequencing joint, and the universal gene detection library of the hereditary familial hypercholesterolemia can be sequenced in the Miseq DX gene sequencer.
In conclusion, the universal gene detection library for hereditary familial hypercholesterolemia prepared by the invention can simultaneously meet Ion GeneStaudioTMThe sequencing requirements of the S5 Plus platform and the Miseq DX platform are met, namely the requirements of two mainstream sequencing platforms, namely an Ion Torrent platform and an Illumina platform, are met, so that the universal gene detection library for hereditary familial hypercholesterolemia has universality.
In addition, the principle and the process of sequencing of all models in the Ion Torrent sequencing platform are consistent, so that the method is suitable for Ion GenestudioTMThe library of S5 Plus sequencers can be adapted to Ion Torrent platform other model sequencers, such as PGM, Proton, etc. Similarly, all model sequencing principles and processes in the Illumina sequencing platform are consistent, and the library suitable for the Miseq DX gene sequencer can be suitable for other model sequencers of the Illumina platform, such as MiniSeq and NextSeq. Therefore, it is clear that the universal gene detection library for hereditary familial hypercholesterolemia of the present invention can be applied to all types of Ion Torrent platform and Illumina platformNumber sequencer.
(3) Two-platform sequencing data analysis showed that the above samples were at Ion GenestudioTMThe following consistent mutations were obtained for both the S5 Plus platform and the Miseq DX platform, indicating the accuracy and reliability of the methods of the invention. Specific data are shown in the following table, and the sequencing results of 10 samples.
The foregoing descriptions of specific exemplary embodiments of the present invention have been presented for purposes of illustration and description. It is not intended to limit the invention to the precise form disclosed, and obviously many modifications and variations are possible in light of the above teaching. The exemplary embodiments were chosen and described in order to explain certain principles of the invention and its practical application to enable one skilled in the art to make and use various exemplary embodiments of the invention and various alternatives and modifications as are suited to the particular use contemplated. It is intended that the scope of the invention be defined by the claims and their equivalents.
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<110> Beijing Anzhiyin Biotechnology Ltd
<120> construction method of universal gene detection library for hereditary familial hypercholesterolemia and kit thereof
<130> 2020
<160> 10
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Claims (9)
1. A universal high throughput sequencing linker for hereditary familial hypercholesterolemia, wherein the linker sequence is as follows;
sequence a:
5’-XXXXXXTAGCTGAGTCGGAGACACGCAGGATCGGAAGAGCACGTCTGAACTCCAGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’；
sequence b:
5’-ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTGCGTGTCTCCGACTCAGCTAXXXXXX-3’；
sequence c:
5’-ACACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCCTCTCTATGGGCAGTCGGTGATXXXXXX-3’
sequence d:
5’-XXXXXXATCACCGACTGCCCATAGAGAGGATCGGAAGAGCACGTCTGAACTCCAGTCACXXXXXXATCTCGTATGCCGTCTTCTG-3’
where XXXXXXX represents the sample tag sequence.
2. The universal high-throughput sequencing linker for hereditary familial hypercholesterolemia according to claim 1, wherein the tag sequence is selected from the group consisting of part or all of SEQ ID nos. 1 to 10.
3. A kit for the construction of a universal genetic testing library for hereditary familial hypercholesterolemia, said kit comprising the universal high throughput sequencing linker of any one of claims 1-2.
4. The kit for the universal gene detection library construction for hereditary familial hypercholesterolemia of claim 3, further comprising a PCR amplification primer set designed for the full coding region and/or variable cleavage region of part or all of the genes LDLR, APOB, PCSK9 and LDLRAP 1.
5. The kit for the construction of the universal gene testing library for hereditary familial hypercholesterolemia according to claim 4, wherein the primer sequence is selected from a part or all of the primer sequences as shown in the following table:
6. use of the universal high throughput sequencing adapter for hereditary familial hypercholesterolemia of any of claims 1-2 in library construction or gene sequencing.
7. A method for constructing a universal gene detection library for hereditary familial hypercholesterolemia, which comprises the following steps:
1) multiplex PCR amplification: designing a synthetic FH primer pool, and performing multiplex PCR amplification on a target region;
2) and (3) digesting a primer sequence: carrying out digestion reaction on the amplification product obtained in the step 1);
3) universal linker preparation, preparing a universal high throughput sequencing linker according to any one of claims 1-2;
4) connecting a sequencing joint: connecting the products of the step 2) and the step 3) under the action of DNA ligase;
5) library purification and amplification: adding magnetic beads into the ligation products obtained in the step 4) for purification, and carrying out PCR amplification on the purified products to obtain the universal gene detection library for hereditary familial hypercholesterolemia.
8. The method for constructing a universal gene test library for hereditary familial hypercholesterolemia according to claim 7, wherein the step 1) comprises designing a synthetic primer pool against all coding regions and/or variable splicing regions of partial or genes of LDLR, APOB, PCSK9 and LDLRAP 1.
9. A universal gene testing library for hereditary familial hypercholesterolemia, which is prepared by the construction method according to any one of claims 7 to 8.
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| CN113684269B (en) * | 2021-08-19 | 2023-03-14 | 江苏百世诺医疗科技有限公司 | Familial hypercholesterolemia gene detection library, construction method and kit thereof |
| CN113684270B (en) * | 2021-08-19 | 2023-03-17 | 江苏百世诺医疗科技有限公司 | Multiple-target capture kit for familial hypercholesterolemia and detection method thereof |
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