CN115323048A - Primer combination and method for detecting human embryo alpha-thalassemia gene mutation - Google Patents
Primer combination and method for detecting human embryo alpha-thalassemia gene mutation Download PDFInfo
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Abstract
The invention provides a primer combination and a method for detecting human embryo alpha-thalassemia gene mutation. The kit consists of primers (1 pair) for specifically amplifying three most common deletion type mutations (20 pairs) of SEA, 4.2kb and 3.7kb of Alpha-thalassemia genes of Chinese population and three most common point mutations of WS, QS and CS, and primers (65 pairs) for specifically amplifying SNP locus regions (including Alpha-globin genes HBA1 and HBA 2) linked with Alpha deletion regions and Single Nucleotide Polymorphism (SNP) loci (65 pairs) closely linked in the range of 1-2Mb at the downstream of the HBA genes, wherein the total number of the primers is 86. The invention also provides a detection method for detecting by using the 86-pair primer combination based on a high-throughput sequencing platform. The invention does not need to carry out full amplification on the target region, and simultaneously the SNP analysis of the corresponding region can directly judge the deletion type of the embryo.
Description
Technical Field
The invention relates to the field of gene detection, in particular to a primer combination and a method for detecting human embryo alpha-thalassemia gene mutation.
Background
Thalassemia is a common group of inherited human hemoglobin diseases that severely threaten human health. Hemoglobin diseases include thalassemia (e.g., α -and β -thalassemia) caused by globin synthesis disorder and abnormal hemoglobin (e.g., hbC, hbE, etc.) caused by structural abnormality, which are the first identified molecular diseases in the world. Some of the early cases of hereditary anemia are derived from immigration in the Mediterranean region, and this group of diseases is called thalassemia (thalassemia), or simply thalassemia. Thalassemia is the most common genetic disease with the greatest harm in southern provinces of China, and the genetic hemolytic hemoglobinopathy which causes reduction or deletion of peptide chain synthesis due to globin gene defect is classified into alpha-thalassemia and beta-thalassemia. Clinically, they are classified into mild (carriers of thalassemia), intermediate and severe types according to their clinical manifestations. At present, 17 deletion mutations are found in China in alpha-thalassemia, and the most common deletion types comprise SEA, 4.2kb and 3.7kb and three point mutations of CS, WS and QS. At present, the alpha-thalassemia is not yet effective in radical treatment, and the prevention is mainly given. Therefore, early detection of carriers of aberrant genes of thalassemia is of great importance for the guidance of prenatal and prenatal diagnosis.
Since α -thalassemia is hereditary hemolytic anemia caused by a defect in the α -globin gene or by partial or complete inhibition of α -globin chain synthesis, most α -thalassemia is caused by a deletion of the α -gene, the α -gene can be examined using genetic diagnosis. The genetic diagnosis of the thalassemia in China starts in the 80 th 20 th century, and successively goes through 5 development stages of DNA dot hybridization, restriction enzyme zymogram analysis, restriction Fragment Length Polymorphism (RFLP) linkage analysis, oligonucleotide (ASO) probe hybridization, PCR in vitro gene amplification and the like, particularly PCR and related development technologies thereof, and the genetic diagnosis method becomes the most common genetic diagnosis method. At present, the clinical pre-implantation genetic diagnosis technology for monogenic genetic diseases can be divided into the traditional PCR (polymerase chain reaction) detection method, the Karyomapping technology based on SNP (single nucleotide polymorphism) chip and the new generation sequencing-target capture sequencing.
Although the traditional PCR is sensitive and can directly judge the condition of a mutation site, when the traditional PCR is applied to genetic diagnosis before embryo implantation, the defects of exogenous pollution, allele Dropout (ADO), amplification failure or dominant amplification and the like exist. However, because the sites of the pathogenic genes of the monogenic genetic diseases are rare, almost no pathogenic gene sites are in the probe detection range of the SNP chip, so that the genotype of the pathogenic sites cannot be directly judged through the SNP chip, and the pathogenic sites can be determined only by the SNP site information at the upstream and the downstream through the family SNP haplotype linkage analysis.
Disclosure of Invention
In order to overcome the technical defects, the invention provides a primer combination and a method for detecting human embryo alpha-thalassemia gene mutation. A primer composition for detecting Alpha-thalassemia gene mutation comprises primers (1 pair) for specifically amplifying three most common deletion type mutations (20 pairs) of Alpha-thalassemia genes of Chinese population, namely SEA, 4.2kb and 3.7kb, and three most common point mutations of WS, QS and CS, and primers (65 pairs) for specifically amplifying Single Nucleotide Polymorphism (SNP) sites which are linked with an Alpha deletion region (comprising Alpha-globin genes HBA1 and HBA 2) and are closely linked within a range of 1-2Mb at the downstream of the HBA genes, wherein the total number of the primers is 86. The invention also provides a method for detecting the human embryo alpha-thalassemia gene mutation, which uses the 86 pairs of primer combination and carries out detection based on a high-throughput sequencing platform.
The multiplex PCR amplification primers for alpha-thalassemia gene mutation detection sequentially comprise: SEA: 1-86, and the specific primer pairs are as follows:
the SNP site information is as follows:
the primer conditions were as follows:
in order to implement the present invention, we have adopted the following techniques and methods:
the pretreatment of the sample adopts a single cell whole genome amplification technology, and the genome of trace cells can be amplified to reach a detectable amount. Firstly, special random primers are randomly combined and extended with a genome, the tail ends of the primers are provided with a special universal sequence, so that the tail ends of amplicons can be complemented, the amplicons form rings by themselves, excessive DNA copying is prevented, amplification products cannot be used as a new template, exponential amplification after DNA copying is prevented to a great extent, and the uniformity of whole genome amplification is increased; the pre-amplified product is exponentially amplified by a pair of primers to increase the yield of the amplified product. When building a library, the amplified whole genome product is subjected to end filling, an A joint is added, and a universal joint for sequencing, a sequencing primer joint and the like are added, so that a constructed sequencing library can be used for on-computer sequencing. Before the on-machine sequencing, performing water-in-oil amplification on the library by adopting an emulsion PCR technology to generate a template library for sequencing, enriching a positive template to meet the total amount requirement of the on-machine sequencing, and finally performing sequencing based on a semiconductor sequencing technology.
The targeted capture method comprises the following steps: the multiplex PCR capture sequencing is a rapid detection mode aiming at clinical and disease gene hot spots, and can be used for carrying out rapid targeted linear amplification on thousands or even tens of thousands of sites such as SNP (single nucleotide polymorphism) and InDel (internal amplified polymorphism) on the premise of ensuring the amplification uniformity, and then carrying out mass sample detection and deep analysis by using a sequencing platform. The invention discloses a primer composition for detecting Alpha-thalassemia gene mutation, which consists of primers (1 pair) for specifically amplifying three most common deletion type mutations (20 pairs) of Alpha-thalassemia genes of Chinese population, namely SEA, 4.2kb and 3.7kb and three most common point mutations of WS, QS and CS, and primers (65 pairs) for specifically amplifying SNP locus regions linked with Alpha deletion regions (comprising Alpha-globin genes HBA1 and HBA 2) and Single Nucleotide Polymorphisms (SNP) loci tightly linked within 1-2Mb downstream of the HBA genes, wherein the total number of the primers is 86. The screening criteria for the primers were: specific region amplification results corresponding to different deletion types, and SNP analysis of corresponding regions can directly judge the type of the embryo; and (3) screening and labeling of linked SNP loci: genomAD, east asain MAF (0.2,0.8), GC content 0.3-0.6, and 1-2M interval upstream and downstream from the detection site of linkage analysis.
High throughput sequencing method: mainly comprises the steps of using a designed multiple primer Panel, and identifying a specific bar code and a DNA library; performing pre-computer quality inspection on the successfully constructed library, homogenizing the library passing the quality inspection, performing template preparation and template enrichment, performing template amplification, and finally performing a sequencing experiment on the positive template on an Ion Torrent sequencing platform.
The biological information analysis method comprises the following steps:
1. the off-line data analysis method comprises the following steps: and (3) carrying out high-throughput sequencing on the prepared sequencing database system in an Ion Torrent Proton platform, and after sequencing is finished, preprocessing the generated original sequencing off-line data by adopting Torrent _ Server _5.0 \ VM software, thereby removing a linker sequence in the off-line data. And aligning the processed sequencing data to GRCh37 (hg 19) human reference genome using a Tmap alignment tool.
2. The method for detecting the gene type of the thalassemia gene comprises the following steps: the preprocessed sequencing data are subjected to genotype Analysis through inserts such as "Coverage Analysis and" Variant Caller "in a Torrent Suite (v5.4.0) Analysis kit, and the Coverage depth and the genotype condition of the haplotype polymorphic Site (SNP) of each gene site in the target region chr16:105290-2154894 are analyzed. For the sample with positive mutation, the table generated by the Variant Caller plug-in is checked, and whether the site is heterozygous mutation or homozygous mutation is further judged according to the mutation frequency in the table (the mutation frequency is heterozygous mutation within the range of 40-60%, and is homozygous mutation within the range of 90-100%). The haplotype is constructed by utilizing the position and the genotype of parents and priors on the chromosome of the SNP closely linked with the pathogenic gene HBA1 of the alpha-thalassemia. And judging whether the embryo is pathogenic or not through the established haplotype.
3. Target region copy number analysis method: a benchmark database was constructed using known normal samples, the relative sequence value (RC) of the SEA deletion region chromosomes was calculated for each sample, and the range of reference values was determined by statistical analysis using the average of the different samples. Next, a deletion judgment is made for each sample by using the reference value.
4. The method for judging the pathogenicity of the embryo by the SNP locus of the deletion region comprises the following steps: and (3) selecting SNP sites which are different from parents in the SEA deletion region, and judging the pathogenicity of offspring according to Mendelian genetic rules.
The invention has the beneficial technical effects that: the target region does not need to be amplified completely, for example, specific SNP sites are selected from the deletion type region for amplification, amplification results of specific regions corresponding to different deletion types are obtained, and the deletion type of the embryo can be directly judged by SNP analysis of the corresponding region. Meanwhile, in the case of deletion of the proband, the result of the embryo is directly used as the proband for analysis (analysis of the deletion-specific interval can directly determine the phenotype of the embryo); the upstream and downstream regions of SEA have certain probability of risk of homologous recombination, and the direct judgment result of embryo can be used as the verification of linkage analysis result.
Drawings
FIG. 1 is a schematic diagram of SNP haplotype technology.
FIG. 2 is the SEA region coverage copy number, indicating that the sample is a homozygous deletion.
Fig. 3 shows the SEA area overlay copy number.
Fig. 4 shows the SEA area overlay copy number.
Fig. 5 shows the SEA area overlay copy number.
FIG. 6 shows the haplotype results, which can be distinguished by color, and M2 and F2 are deletion chains.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings of the present invention.
The first embodiment is as follows: 4 cases of embryo alpha-thalassemia pre-transplantation detection based on Ion Torrent sequencing platform
The method comprises the following main experimental steps:
(1) Peripheral blood DNA extraction
In this example, 2 samples of α -thalassemia-SEA/α parents were obtained, and samples from both couples were obtained from peripheral Blood samples, and extracted using Magpure Fast Blood DNA LQ Kit according to the Kit instructions.
(2) Whole genome amplification
In this example, 4 embryo biopsy samples of this family were obtained, and the 4 embryo samples were subjected to whole genome amplification according to the Fapon single Cell gene-amplification Kit standard procedure.
(3) And (3) carrying out the concentration determination of the Qubit4.0 on the sample DNA obtained in the step (1) and (2).
In this example, the DNA sample was assayed for concentration using the dsDNA HS Assay Kit, and the detection was performed according to the Kit instructions.
(4) Multiplex PCR amplification: adding reagents into the quantified products according to a multiplex amplification system to perform multiplex PCR amplification according to the following system:
| reagent (reagent) | Volume Per reaction(μl) |
| 1 mu LWGA template or 30ngDN template | 1μL/ |
| 5xHyper Buffer | |
| 2 | |
| MultiPrimer DNA Polymerase | 0.25 |
| Multiplex primer Mix | 0.34 |
| Nuclease-Free Water | 6.41 |
| Total volume of the |
10 |
Reaction conditions are as follows:
quality inspection of multiple amplification products: 1.5 mu L of the amplified product is taken for electrophoresis quality detection, the concentration of agarose gel (2%) confirms that the target fragment is successfully amplified.
And (3) purifying an amplification product: purifying an amplification product by magnetic Beads, wherein in the step, DNA Clean Beads Kit is adopted, and the sample: magnetic beads = 1.2;
purified PCR product, concentration determination using qubit4.0
(5) Library construction: the Library construction Kit adopted in the Step is Fapon Two-Step Library Prep Kit for Ion Torrent
a end filling
The end filling reaction system is a 50 mu L system, and the components of the reaction system are as follows:
| reagent (reagent) | Volume Per reaction(μl) |
| DNA/>50ng | X |
| |
10 |
| End |
5 |
| Nuclease-Free-Water | 35-X |
| Total | 50 |
b connection of joints
The P1 linker used in this step is:
CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG-s-T-s-T
the adapter linker (a known short nucleotide sequence used for linking unknown target sequencing fragments) used in this step is 96 Barcode x consisting of the following positive and negative sequences, and the specific sequences represented by Barcode x are as follows:
the joint connection reaction system is a 100 mu L system, and the components of the reaction system are as follows:
| reagent (reagent) | Volume Per reaction(μl) |
| End-filling product | 50 |
| Ligation Buffer | 34 |
| |
6 |
| |
1 |
| Nuclease-Free- |
8 |
| |
1 |
| Total | 100 |
The adaptor ligation PCR reaction conditions were as follows: 15min at 20 ℃; hold at 4 DEG C
c linker ligation product purification
In the step, a DNA Clean Beads Kit is adopted, and the sample: magnetic bead = 1.9; 25 μ L of adaptor-ligated library fragments were obtained.
d PCR amplification of library fragments
The sequence of the library amplification primers is as follows:
Primer-1 CCACTACGCCTCCGCTTTCCTCTCTATG
Primer-2 CCATCTCATCCCTGCGTGTC
the amplification reaction system is a 50 mu L system, and the components of the reaction system are as follows:
| reagent (reagent) | Volume Per reaction(μl) |
| Purified ligation product | 50 |
| 2×HIFI Seq PCR Mix | 34 |
| |
6 |
| Total | 90 |
The PCR amplification reaction conditions were as follows: 10min at 72 ℃; 6 cycles at 98 ℃ for 45s, (98 ℃ for 20s,65 ℃ for 30s, and 72 ℃ for 30 s); 5min at 72 ℃; hold at 4 deg.C
e purification of amplification product
In the step, a DNA Clean Beads Kit is adopted, and the sample: magnetic beads =1:1; 25 μ L of adaptor-ligated library fragments were obtained.
The DNA library obtained in the step is subjected to concentration determination by adopting a1 x dsDNA HS Assay Kit, and the detection is carried out according to the operation steps of the Kit specification.
(6) Preparing a template: library products were homogenized and mixed to 100pM
1) The ng/ul concentration was converted to nM concentration as follows:
(concentration in ng/μl)×10 6 ÷(660g/mol×average library size)
＝concentration in nM
2) Sampling was performed according to the following formula
Volume of samples＝V(f)*C(f)/#*C(i)
Note: wherein the content of the first and second substances,
v (f): final volume of sample after mixing;
c (f): final concentration of all samples after mixing;
#: number of all samples of Mix;
c (i): initial concentration (nM) of each sample;
final volume was made up with EB.
The standard operation of the Ion P1 Hi-Q OT2 kit is followed
Illustrating template preparation and template enrichment.
(7) And (3) machine sequencing: and performing on-machine sequencing according to the standard operation instruction of the Ion P1 Hi-Q sequencing 200 kit.
(II) data analysis: a benchmark database was constructed using known normal samples, the relative sequence value (RC) of the SEA deletion region chromosomes was calculated for each sample, and the range of reference values was determined by statistical analysis using the average of the different samples. Next, a deletion judgment is made for each sample by using the reference value.
(III) haplotype analysis
(1) The results of the sample measurements are shown in Table 1,2
(2) The results of the sample copy number analysis are shown in FIG. 2,3,4,5
(3) The results of the sample haplotypes are shown in FIG. 6
Table 1 sample test results show
Table 2: the result of the SNP site of the deletion region shows
The above examples only represent some embodiments of the present invention, and the description thereof is more specific and detailed, but not to be construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention. Therefore, the protection scope of the present invention shall be subject to the claims.
Claims (6)
1. A primer combination for detecting human embryo alpha-thalassemia gene mutation, which is characterized by comprising a primer with the sequence of SEA ID NO: 1-86 of the multiplex PCR primers.
2. The primer combination for detecting human embryonic Alpha-thalassemia gene mutation according to claim 1, which comprises 20 pairs of primers for specifically amplifying SEA, three deletion type mutations of 4.2kb and 3.7kb of Alpha-thalassemia gene of human population, 1 pair of primers for three point mutations of WS, QS and CS, and 65 pairs of primers for specifically amplifying linked SNP site region of Alpha-globin gene HBA1 and HBA2 and tightly linked SNP site in 1-2Mb range downstream of HBA gene.
3. A method for detecting human embryonic alpha-thalassemia gene mutation, which comprises the following steps, using the primer combination for detecting human embryonic alpha-thalassemia gene mutation of any one of claims 1 or 2:
the method comprises the following steps: extracting peripheral blood DNA and embryonic cells of couples to be detected to carry out whole genome amplification: extracting DNA from peripheral blood, enriching peripheral blood genome DNA, enriching genome DNA in cells by carrying out single cell whole genome amplification on trophoblast cells in a cleavage stage or a blastocyst stage on the 3 rd to 6 th day of in-vitro culture, and carrying out concentration measurement on the obtained DNA;
step two: amplification of the target fragment: mixing the DNA enriched in the step one with a multiplex PCR amplification reagent and the primer combination, and carrying out multiplex PCR reaction to obtain an amplified target gene DNA fragment;
step three: and (3) purifying an amplification product: carrying out magnetic bead purification on the DNA fragments subjected to the multiple PCR reaction, and removing redundant small fragment joints and redundant primers to obtain a purified multiple product;
step four: filling the tail end in a plain mode: adding a primer capable of being combined with the cohesive end into the DNA fragment obtained in the step three, mixing the primer with an end filling reagent, and incubating to obtain a DNA sample with a blunt end;
step five: connecting a joint: mixing the blunt-end DNA sample obtained in the fourth step with a P1 joint, an adapter joint and a joint connection reaction reagent for a connection reaction to obtain a DNA fragment connected with the joint;
step six: magnetic bead purification: performing magnetic bead purification on the DNA fragments of the connection joints in the step five, and removing redundant small fragment joints and redundant primers to obtain purified DNA fragments of the connection joints;
step seven: library amplification and detection: and adding the DNA fragment of the connecting joint purified in the step six into a library amplification primer and a library amplification reaction reagent for PCR amplification, purifying by adopting magnetic beads to obtain a DNA library of a target fragment, and measuring the concentration of the obtained DNA library.
Step eight: high-throughput sequencing: performing high-throughput sequencing on the DNA library obtained in the step seven by adopting a high-throughput sequencing platform;
step nine: bioinformatics analysis: performing multiple capture sequencing on the whole genome amplification product of the embryo biopsy cells and the family whole blood DNA sample, performing bioinformatics SNP analysis to obtain haplotype information linked with the disease-causing genes of both couples, and judging whether the disease-causing haplotype of parents is inherited according to the sequencing data of the embryo sample.
4. The method for detecting human embryonic α -thalassemia gene mutation of claim 3, wherein the multiple PCR reaction conditions in the second step are as follows:
denaturation: denaturation at 95 ℃ for 10min,1 cycle
Pre-amplification: pre-denaturation: denaturation at 94 ℃ for 30 s;
annealing: annealing at 58 deg.C for 2min, 60 deg.C for 3min, and 62 deg.C for 1min;
extension: extending for 1min at 72 ℃;
annealing + extension for 5 cycles;
enrichment and amplification: denaturation: 94 ℃ for 20s
And (3) annealing: annealing at 66 deg.C for 3min, and performing 25 cycles;
and terminated at 4 ℃.
5. The method for detecting human embryonic α -thalassemia gene mutation of claim 3, wherein the P1 linker sequence in step five is:
CCACTACGCCTCCGCTTTCCTCTCTATGGGCAGTCGGTGAT
ATCACCGACTGCCCATAGAGAGGAAAGCGGAGGCGTAGTGG-s-T-s-T
the adapter joint is 96 Barcode x consisting of the following positive and negative sequences, and the specific sequences represented by the Barcode x are as follows:
A1-index 1 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTAAGGTAACGAT
A1-1 ATCGTTACCTTAGCTGAGTCGGAGACACGC
A1-index 2 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTAAGGAGAACGAT
A2-1 ATCGTTCTCCTTACTGAGTCGGAGACACGC
A1-index 3 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAAGAGGATTCGAT
A3-1 ATCGAATCCTCTTCTGAGTCGGAGACACGC
A1-index 4 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTACCAAGATCGAT
A4-1 ATCGATCTTGGTACTGAGTCGGAGACACGC
A1-index 5 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCAGAAGGAACGAT
A5-1 ATCGTTCCTTCTGCTGAGTCGGAGACACGC
A1-index 6 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTGCAAGTTCGAT
A6-1 ATCGAACTTGCAGCTGAGTCGGAGACACGC
A1-index 7 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCGTGATTCGAT
A7-1 ATCGAATCACGAACTGAGTCGGAGACACGC
A1-index 8 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCCGATAACGAT
A8-1 ATCGTTATCGGAACTGAGTCGGAGACACGC
A1-index 9 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTGAGCGGAACGAT
A9-1 ATCGTTCCGCTCACTGAGTCGGAGACACGC
A1-index 10 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTGACCGAACGAT
A10-1 ATCGTTCGGTCAGCTGAGTCGGAGACACGC
A1-index 11 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTCGAATCGAT
A11-1 ATCGATTCGAGGACTGAGTCGGAGACACGC
A1-index 12 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTAGGTGGTTCGAT
A12-1 ATCGAACCACCTACTGAGTCGGAGACACGC
A1-index 13 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTAACGGACGAT
A13-1 ATCGTCCGTTAGACTGAGTCGGAGACACGC
A1-index 14 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTGGAGTGTCGAT
A14-1 ATCGACACTCCAACTGAGTCGGAGACACGC
A1-index 15 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTAGAGGTCGAT
A15-1 ATCGACCTCTAGACTGAGTCGGAGACACGC
A1-index 16 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTGGATGACGAT
A16-1 ATCGTCATCCAGACTGAGTCGGAGACACGC
A1-index 17 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTATTCGTCGAT
A17-1 ATCGACGAATAGACTGAGTCGGAGACACGC
A1-index 18 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAGGCAATTGCGAT
A18-1 ATCGCAATTGCCTCTGAGTCGGAGACACGC
A1-index 19 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTAGTCGGACGAT
A19-1 ATCGTCCGACTAACTGAGTCGGAGACACGC
A1-index 20 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCAGATCCATCGAT
A20-1 ATCGATGGATCTGCTGAGTCGGAGACACGC
A1-index 21 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCGCAATTACGAT
A21-1 ATCGTAATTGCGACTGAGTCGGAGACACGC
A1-index 22 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCGAGACGCGAT
A22-1 ATCGCGTCTCGAACTGAGTCGGAGACACGC
A1-index 23 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTGCCACGAACGAT
A23-1 ATCGTTCGTGGCACTGAGTCGGAGACACGC
A1-index 24 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAACCTCATTCGAT
A24-1 ATCGAATGAGGTTCTGAGTCGGAGACACGC
A1-index 25 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCTGAGATACGAT
A25-1 ATCGTATCTCAGGCTGAGTCGGAGACACGC
A1-index 26 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTACAACCTCGAT
A26-1 ATCGAGGTTGTAACTGAGTCGGAGACACGC
A1-index 27 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAACCATCCGCGAT
A27-1 ATCGCGGATGGTTCTGAGTCGGAGACACGC
A1-index 28 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGATCCGGAATCGAT
A28-1 ATCGATTCCGGATCTGAGTCGGAGACACGC
A1-index 29 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCGACCACTCGAT
A29-1 ATCGAGTGGTCGACTGAGTCGGAGACACGC
A1-index 30 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGAGGTTATCGAT
A30-1 ATCGATAACCTCGCTGAGTCGGAGACACGC
A1-index 31 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCAAGCTGCGAT
A31-1 ATCGCAGCTTGGACTGAGTCGGAGACACGC
A1-index 32 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTTACACACGAT
A32-1 ATCGTGTGTAAGACTGAGTCGGAGACACGC
A1-index 33 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCTCATTGAACGAT
A33-1 ATCGTTCAATGAGAACTGAGTCGGAGACACGC
A1-index 34 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCGCATCGTTCGAT
A34-1 ATCGAACGATGCGACTGAGTCGGAGACACGC
A1-index 35 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTAAGCCATTGTCGAT
A35-1 ATCGACAATGGCTTACTGAGTCGGAGACACGC
A1-index 36 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAAGGAATCGTCGAT
A36-1 ATCGACGATTCCTTCTGAGTCGGAGACACGC
A1-index 37 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTTGAGAATGTCGAT
A37-1 ATCGACATTCTCAAGCTGAGTCGGAGACACGC
A1-index 38 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTGGAGGACGGACGAT
A38-1 ATCGTCCGTCCTCCACTGAGTCGGAGACACGC
A1-index 39 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTAACAATCGGCGAT
A39-1 ATCGCCGATTGTTACTGAGTCGGAGACACGC
A1-index 40 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTGACATAATCGAT
A40-1 ATCGATTATGTCAGCTGAGTCGGAGACACGC
A1-index 41 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCCACTTCGCGAT
A41-1 ATCGCGAAGTGGAACTGAGTCGGAGACACGC
A1-index 42 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGAGCACGAATCGAT
A42-1 ATCGATTCGTGCTCTGAGTCGGAGACACGC
A1-index 43 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTTGACACCGCGAT
A43-1 ATCGCGGTGTCAAGCTGAGTCGGAGACACGC
A1-index 44 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTGGAGGCCAGCGAT
A44-1 ATCGCTGGCCTCCAACTGAGTCGGAGACACGC
A1-index 45 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTGGAGCTTCCTCGAT
A45-1 ATCGAGGAAGCTCCACTGAGTCGGAGACACGC
A1-index 46 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCAGTCCGAACGAT
A46-1 ATCGTTCGGACTGACTGAGTCGGAGACACGC
A1-index 47 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTAAGGCAACCACGAT
A47-1 ATCGTGGTTGCCTTACTGAGTCGGAGACACGC
A1-index 48 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCTAAGAGACGAT
A48-1 ATCGTCTCTTAGAACTGAGTCGGAGACACGC
A1-index 49 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTAACATAACGAT
A49-1 ATCGTTATGTTAGGACTGAGTCGGAGACACGC
A1-index 50 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGGACAATGGCGAT
A50-1 ATCGCCATTGTCCGCTGAGTCGGAGACACGC
A1-index 51 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTGAGCCTATTCGAT
A51-1 ATCGAATAGGCTCAACTGAGTCGGAGACACGC
A1-index 52 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCGCATGGAACGAT
A52-1 ATCGTTCCATGCGGCTGAGTCGGAGACACGC
A1-index 53 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTGGCAATCCTCGAT
A53-1 ATCGAGGATTGCCAGCTGAGTCGGAGACACGC
A1-index 54 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCGGAGAATCGCGAT
A54-1 ATCGCGATTCTCCGGCTGAGTCGGAGACACGC
A1-index 55 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCACCTCCTCGAT
A55-1 ATCGAGGAGGTGGACTGAGTCGGAGACACGC
A1-index 56 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCAGCATTAATTCGAT
A56-1 ATCGAATTAATGCTGCTGAGTCGGAGACACGC
A1-index 57 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTGGCAACGGCGAT
A57-1 ATCGCCGTTGCCAGACTGAGTCGGAGACACGC
A1-index 58 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTAGAACACGAT
A58-1 ATCGTGTTCTAGGACTGAGTCGGAGACACGC
A1-index 59 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTTGATGTTCGAT
A59-1 ATCGAACATCAAGGACTGAGTCGGAGACACGC
A1-index 60 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTAGCTCTTCGAT
A60-1 ATCGAAGAGCTAGACTGAGTCGGAGACACGC
A1-index 61 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCACTCGGATCGAT
A61-1 ATCGATCCGAGTGACTGAGTCGGAGACACGC
A1-index 62 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCCTGCTTCACGAT
A62-1 ATCGTGAAGCAGGAACTGAGTCGGAGACACGC
A1-index 63 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCTTAGAGTTCGAT
A63-1 ATCGAACTCTAAGGCTGAGTCGGAGACACGC
A1-index 64 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTGAGTTCCGACGAT
A64-1 ATCGTCGGAACTCAGCTGAGTCGGAGACACGC
A1-index 65 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTGGCACATCGAT
A65-1 ATCGATGTGCCAGGACTGAGTCGGAGACACGC
A1-index 66 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCGCAATCATCGAT
A66-1 ATCGATGATTGCGGCTGAGTCGGAGACACGC
A1-index 67 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCCTACCAGTCGAT
A67-1 ATCGACTGGTAGGAACTGAGTCGGAGACACGC
A1-index 68 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCAAGAAGTTCGAT
A68-1 ATCGAACTTCTTGACTGAGTCGGAGACACGC
A1-index 69 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTCAATTGGCGAT
A69-1 ATCGCCAATTGAACTGAGTCGGAGACACGC
A1-index 70 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCTACTGGTCGAT
A70-1 ATCGACCAGTAGGCTGAGTCGGAGACACGC
A1-index 71 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTGAGGCTCCGACGAT
A71-1 ATCGTCGGAGCCTCACTGAGTCGGAGACACGC
A1-index 72 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGAAGGCCACACGAT
A72-1 ATCGTGTGGCCTTCGCTGAGTCGGAGACACGC
A1-index 73 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCTGCCTGTCGAT
A73-1 ATCGACAGGCAGACTGAGTCGGAGACACGC
A1-index 74 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGATCGGTTCGAT
A74-1 ATCGAACCGATCGCTGAGTCGGAGACACGC
A1-index 75 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCAGGAATACGAT
A75-1 ATCGTATTCCTGACTGAGTCGGAGACACGC
A1-index 76 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGGAAGAACCTCGAT
A76-1 ATCGAGGTTCTTCCGCTGAGTCGGAGACACGC
A1-index 77 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGAAGCGATTCGAT
A77-1 ATCGAATCGCTTCGCTGAGTCGGAGACACGC
A1-index 78 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCAGCCAATTCTCGAT
A78-1 ATCGAGAATTGGCTGCTGAGTCGGAGACACGC
A1-index 79 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCTGGTTGTCGAT
A79-1 ATCGACAACCAGGCTGAGTCGGAGACACGC
A1-index 80 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCGAAGGCAGGCGAT
A80-1 ATCGCCTGCCTTCGACTGAGTCGGAGACACGC
A1-index 81 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCTGCCATTCGCGAT
A81-1 ATCGCGAATGGCAGGCTGAGTCGGAGACACGC
A1-index 82 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTGGCATCTCGAT
A82-1 ATCGAGATGCCAACTGAGTCGGAGACACGC
A1-index 83 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTAGGACATTCGAT
A83-1 ATCGAATGTCCTAGCTGAGTCGGAGACACGC
A1-index 84 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTTCCATAACGAT
A84-1 ATCGTTATGGAAGCTGAGTCGGAGACACGC
A1-index 85 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCAGCCTCAACGAT
A85-1 ATCGTTGAGGCTGGCTGAGTCGGAGACACGC
A1-index 86 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTTGGTTATTCGAT
A86-1 ATCGAATAACCAAGCTGAGTCGGAGACACGC
A1-index 87 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTGGCTGGACGAT
A87-1 ATCGTCCAGCCAACTGAGTCGGAGACACGC
A1-index 88 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCCGAACACTTCGAT
A88-1 ATCGAAGTGTTCGGCTGAGTCGGAGACACGC
A1-index 89 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCTGAATCTCGAT
A89-1 ATCGAGATTCAGGACTGAGTCGGAGACACGC
A1-index 90 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTAACCACGGCGAT
A90-1 ATCGCCGTGGTTAGCTGAGTCGGAGACACGC
A1-index 91 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGGAAGGATGCGAT
A91-1 ATCGCATCCTTCCGCTGAGTCGGAGACACGC
A1-index 92 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTAGGAACCGCGAT
A92-1 ATCGCGGTTCCTAGCTGAGTCGGAGACACGC
A1-index 93 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCTTGTCCAATCGAT
A93-1 ATCGATTGGACAAGCTGAGTCGGAGACACGC
A1-index 94 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTCCGACAAGCGAT
A94-1 ATCGCTTGTCGGACTGAGTCGGAGACACGC
A1-index 95 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGCGGACAGATCGAT
A95-1 ATCGATCTGTCCGCTGAGTCGGAGACACGC
A1-index 96 CCATCTCATCCCT-s-G-s-CGTGTCTCCGACTCAGTTAAGCGGTCGAT
A96-1 ATCGACCGCTTAACTGAGTCGGAGACACGC
6. the method for detecting human embryonic α -thalassemia gene mutation of claim 3, wherein the library amplification primer sequence of step seven is:
Primer-1 CCACTACGCCTCCGCTTTCCTCTCTATG
Primer-2 CCATCTCATCCCTGCGTGTC
the PCR amplification reaction conditions are as follows:
and (3) notching and filling in: 10min at 72 ℃;
denaturation: the temperature of the mixture is 45s at 98 ℃,
annealing: 6 cycles of 98 ℃ for 20s,65 ℃ for 30s and 72 ℃ for 30 s;
extension: 5min at 72 ℃;
terminating at 4 ℃.
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