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  • C12Q2600/156—Polymorphic or mutational markers

Definitions

• the present invention belongs to the field of genomic bioinformatics analysis, and in particular, to a method for typing thalassemia and its use. Background technique
• Thalassemia is a common autosomal recessive disorder. It is caused by globin gene variation, impaired globin biosynthesis, and insufficient or absent production. Thalassemia is extremely high in all provinces south of the Yangtze River in China, and it is one of the most influential genetic diseases in China.
• peptide chains constituting globin There are four kinds of peptide chains constituting globin, namely ⁇ , , and 5 chains, which are respectively encoded by their corresponding genes. Deletion or point mutation of these genes can cause structural disorder of the peptide chain, resulting in changes in the composition of hemoglobin.
• the two most common types are: a thalassemia due to alpha chain variation and beta thalassemia due to beta chain variation.
• the ⁇ -globin gene is located in the ⁇ -globin gene cluster at the end of the short arm of chromosome 16, which consists of two repeated ⁇ genes ( ⁇ 2 and al), one embryonic a gene ( ⁇ 2), and three pseudogenes. ( ⁇ ⁇ , ⁇ 2 and ⁇ ) and a functionally unknown gene ( ⁇ 1), the order of which is: 5 '-2 ⁇ 22 ⁇ 12 ⁇ 22 ⁇ 12 ⁇ 22 ⁇ 12 ⁇ 12-3 ', and the total length is about 30kb.
• the ⁇ -globin gene is located in the short arm of the chromosome 1 (1 1 ⁇ 15), and the gene sequence is 5'-2 ⁇ 20 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2 ⁇ 2-3', and the length is about 60kb.
• the classification of thalassemia is the detection of mutations in ⁇ -chain and ⁇ -chain related genes, including point mutations, insertion deletions and copy number variations.
• There are many methods for detecting thalassemia including reverse dot blot hybridization, PCR2 single-strand conformation polymorphism analysis, and gene chip method.
• Reverse dot blotting is a new technique for detecting point mutations. It replaces the immobilized target DNA with a fixed probe on the membrane, thus changing the traditional hybridization method to detect only one mutation at a time. the way. The membrane strips from which a plurality of specific probes are immobilized are hybridized with the amplified target sequence, so that one hybridization can be simultaneously detected and detected. However, reverse dot hybridization can only detect partial point mutations and is not universal.
• PC 2 single strand conformation polymerase is based on the principle that the DNA single strand can be electrophoresed on a neutral polyacrylamide gel even if only one base changes. The mobility has changed.
• the process is as follows: First, the target DNA fragment is amplified by PCR, and then the PCR product is denatured at 95 ° C and then subjected to neutral polyacrylamide gel. Electrophoresis, DNA electrophoresis bands can be displayed by methods such as radionuclide and silver staining. Because silver staining is simple and easy, and there is no radionuclide contamination, it has become the preferred method for displaying SSCP electrophoresis bands. However, this method is slow in typing and high in cost, and is not suitable for popularization and application.
• Affymetrix began to use DNA chip technology to diagnose thalassemia.
• the basic principle is to simultaneously hybridize with the same cDNA chip with two different fluorescent dye-labeled target sequences, and analyze the fluorescence signal intensity of different colors. It can reflect changes in gene expression. Since this technology can immobilize an extremely large number of probes on the support at the same time, a large number of biomolecules can be detected and analyzed at one time, thereby solving the single defect of the detection items such as conventional PCR and nucleic acid blot hybridization, but the method exists. Low throughput, low cost and other shortcomings.
• Another object of the present invention is to provide a technique for detecting SNP and Indel mutations and an application thereof.
• Another object of the present invention is to provide a method for detecting copy number variation and an application thereof.
• Another object of the present invention is to provide a thalassemia typing technique and its use.
• a method of detecting a copy number variation of a HBB gene of a sample to be tested comprising the steps of:
• step (1) Correcting and/or counting the reading sequence obtained in step (1), obtaining the following absolute data values: reading number of the HBB gene of the sample to be tested, reading number of the control gene of the sample to be tested, reading of the normal sample HBB gene Ordinal number, the number of readings of normal sample control genes;
• the HBB gene of the sample to be tested is sequenced using a high throughput sequencing technique to obtain a gene sequence.
• the length of the HBB gene sequence of the sample to be tested obtained in (1) is 70-100 bp. It is preferably 80-90 bp, more preferably 87 bp.
• the HBB gene sequence of the sample to be tested corresponds to the sample and/or gene using the index and the specificity of the primer.
• the reading order obtained by the step (1) is corrected based on the SNP and/or InDel.
• a method of detecting a copy number variation of a HBA gene of a sample to be tested comprising the steps of:
• step (1) Correcting and/or counting the reading order obtained in step (1), obtaining the following absolute data values: the reading number of the normal sample HBA1 gene, the reading number of the normal sample HBA2 gene, the reading number of the normal sample control gene, The number of readings of the HBA1 gene to be tested, the number of readings of the HBA2 gene to be tested, and the number of readings of the control gene to be tested;
• the reading number of the normal sample HBA1 gene/the reading number of the normal sample control gene The number of readings of the HBA2 gene to be tested The number of readings of the HBA2 gene/the number of readings of the test sample to be tested.
• the HBA1 gene or HBA2 gene sequence of the sample to be tested obtained in (1) has a sequence length of 100-150 bp, preferably 120-140 bp, more preferably 128 bp.
• the HBA1 gene or HBA2 gene of the sample to be tested is sequenced using a high-throughput sequencing technique to obtain a gene sequence.
• the HBA1 gene or HBA2 gene of the sample to be tested is associated with the sample and the gene using the index and the specificity of the primer.
• the reading order obtained by the step (1) is corrected according to the SNP and/or InDel.
• the (4) includes the steps of:
• a detecting unit for detecting a copy number variation of a HBB gene of a sample to be tested, the unit comprising a module:
• a sequence acquisition module configured to obtain a HBB gene sequence of the sample to be tested, the sequence corresponding to the sample and/or the gene;
• a read order correction and/or statistics module for correcting and/or counting the read order obtained by the module (1);
• An output module for outputting information on the copy number variation of the HBB gene of the sample to be tested.
• a detecting unit for detecting a copy number variation of a HBA gene of a sample to be tested, the unit comprising a module:
• a sequence acquisition module configured to obtain an HBA1 gene sequence and an HBA2 gene sequence of the sample to be tested, the sequence corresponding to the sample and/or the gene;
• An output module for outputting HBA gene copy number variation information of the sample to be tested.
• a typing system for detecting thalassemia comprising:
• system further comprises: (3) a detection unit of the SNP and/or InDel.
• a method for detecting SNP and Indel variation of a sample to be tested comprising the steps of:
• sequencing data in (1) is aligned with a reference sequence using an alignment software selected from the group consisting of BWA, SOAP, BLAST, MAQ, and the like.
• the sample to be tested is a sample of a person.
• the reference sequence is from hgl9.
• a method of Mediterranean typing of a sample to be tested comprising the steps of:
• step (2) Combine all the variation data of step (1) to obtain the complete variation of the sample to be tested, and then perform the Mediterranean classification of the sample to be tested.
• sample SNP and InDel variation data to be tested are obtained by the method described in the sixth aspect.
• the HBB gene copy number variation of the sample to be tested is detected by the method described in the first aspect.
• the HBA gene copy number variation of the sample to be tested is detected by the method described in the second aspect.
• a method of constructing a thalassemia type SNP and InDel relational database comprising the steps of:
• a SNP and Indel database constructed using the method of the eighth aspect. It is to be understood that within the scope of the present invention, the various technical features of the present invention and the technical features specifically described hereinafter (as in the embodiments) may be combined with each other to constitute a new or preferred technical solution. Due to space limitations, we will not repeat them here. DRAWINGS
• Figure 1 shows the SNP and InDel information analysis process for thalassemia.
• FIG. 2 shows the HBB gene copy number detection information flow.
• FIG. 3 shows the flow of HBA gene copy number information analysis.
• Figure 4 shows the design of the primer.
• Figure 5 shows a single site depth profile.
• the inventors have conducted extensive and intensive research to provide a method for detecting the copy number variation of the HBB gene and/or HBA gene of a sample to be tested, the method comprising the steps of: obtaining a sequence of a gene of interest, the sequence and the sample and/or Corresponding to the gene; correcting and/or counting the reading sequence to obtain absolute data values of related genes; calculating A value or calculating R1 value, R2 value, R3 value; according to A value or R1 value, R2 value, R3 value Obtain information on the copy number variation of the sample HBB and/or HBA gene to be tested.
• the present invention also provides a typing system and analytical method for detecting thalassemia. The present invention has been completed on this basis. the term
• SNP single nucleotide polymorphism
• Polymorphisms expressed by SNPs involve only a single base variation, which can be caused by a single base transition or transversion, or by the insertion or deletion of a base.
• InDel and "insertion deletion mutation” are used interchangeably and refer to mutations involving nucleoside insertions and/or deletions.
• CNV copy number variation
• SNV DNA fragments. Segment size ranges from kb to Mb submicroscopic mutations. As a variation within a chromosome, CNV means that there are too many or insufficient DNA fragments in a certain cell. Double-end sequencing
• the gene fragments (including DNA and cDNA) are sequenced, and the sequenced objects are a piece of physically continuous base sequence called an insert, the length of which is called the insert size.
• double-end sequencing is the sequencing of the base sequences on both sides of the fragment from edge to interior.
• the sequence measured is called read and the length is called read-length.
• the read order measured on both sides is from the same insert, and the distance between the ends is insertsize, so the pairing relationship of the read order on both sides is determined. These two readings are called Pair-end reads.
• High-throughput sequencing of the genome enables humans to detect abnormal changes in disease-associated genes as early as possible, and to facilitate in-depth research into the diagnosis and treatment of individual diseases.
• Those skilled in the art can generally perform high throughput sequencing using conventional methods including, but not limited to, 454 FLX (Roche), Solexa Genome Analyzer (Illumina), Applied Biosystems, SOLID, and the like.
• the common feature of these platforms is the extremely high sequencing throughput.
• high-throughput sequencing can read 400,000 to 4 million sequences in one experiment. The read length is from 25bp depending on the platform. Up to 450 bp, so different sequencing platforms can read base numbers ranging from 1G to 14G in one experiment.
• Solexa high-throughput sequencing includes two steps: DNA cluster formation and on-machine sequencing: a mixture of PCR amplification products is hybridized with a fixed sequencing probe immobilized on a solid phase carrier, and subjected to solid phase bridge PCR amplification to form a sequencing cluster; The sequencing cluster is sequenced by "edge synthesis-edge sequencing” to obtain a sequence of nucleic acid molecules in the sample.
• the DNA cluster is formed by using a flow cell with a single-stranded primer attached to the surface, and the DNA fragment of the single-stranded state is immobilized on the chip by the principle that the linker sequence and the primer on the surface of the chip are complementary to each other by base complementation.
• the fixed single-stranded DNA becomes double-stranded DNA
• the double strand is denatured into a single strand, one end of which is anchored on the sequencing chip, and the other end is randomly and adjacent to another primer to be anchored, Forming a "bridge"
• on the sequencing chip there are tens of millions of DNA single molecules simultaneously reacting; forming a single-stranded bridge, using the surrounding primers as amplification primers, and amplifying again on the surface of the amplification chip to form a double
• the chain the double strand is denatured into a single strand, and becomes a bridge again.
• the template called the next round of amplification continues to expand; after repeated rounds of 30 rounds of amplification, each single molecule is amplified 1000 times, called a single gram. Long DNA cluster.
• DNA clusters were sequenced on a Solexa sequencer. During the sequencing reaction, the four bases were labeled with different fluorescence, and each base was blocked by a protected base. Only one base could be added to a single reaction. After reading the color of the reaction, the protection group is removed, and the next reaction can be continued. Thus, the exact sequence of the base is obtained. In the Solexa Multiplexed Sequencing process, Index is used to distinguish the samples, and after the conventional sequencing is completed, the Index part is additionally sequenced. By index identification, a plurality of different sequencing channels can be distinguished. sample. Thalassemia type SNP&InDel database
• the invention provides a thalassemia type SNP&InDel database and a construction method thereof.
• the contents of the database are as follows:
• the first column is the relative position of the mutation
• the second column is the specific mutation information: as the first row HBA1 represents the gene, A>G indicates that the reference sequence is abrupt from G to G, and the last column represents the type, indicating HBA1
• the 71 bp position of the gene was changed from A to G, and the thalassemia type was Initiation codon (A -> G).
• the type of the database can be supplemented.
• the reference sequence of position 542 of HBA1 has G to T, which is a synonymous mutation, and the last type gives Normal.
• the present invention also provides a method for constructing a thalassemia type-SNP&InDel relational database.
• the method comprises the steps of: a) selecting a sequence of a plurality of normal disease-free genes as a reference sequence (such as a sequence corresponding to coordinates in hgl9) Extracted as a thalassemia reference sequence); Thalassemia-related genes include HBB, HBA 1, HBA2; b) Align the known types of HB A 1 gene, HBA2 gene and HBB gene in the existing thalassemia database with reference sequences The difference sites of the reference sequence, namely the SNP locus and the InDel locus, were obtained, and the SNP and InDel relationship of each type with respect to the reference sequence were obtained, and the thalassemia type SNP-InDel database was obtained.
• the invention provides a method for detecting SNP and Indel mutations, comprising the steps of: sequencing data
• the SNP&InDel linkage relationship with respect to the reference sequence is obtained by comparison with the corresponding reference sequence, and the type is determined by the SNP and InDel linkage relationship.
• the sample sequence is compared with the reference sequence by using comparison software (preferably BWA, SOAP, BLAST, MAQ, etc.); then the comparison result file is format converted (preferably samtools software tool is converted), and the consistency is obtained. Sexual sequence file; Finally, compared with the SNP&InDel linkage database, the type results of thalassemia were obtained.
• comparison software preferably BWA, SOAP, BLAST, MAQ, etc.
• the SNP&InDel mutation detection method comprises the steps of:
• the obtained SNP and InDel are annotated; then the SNP and InDel are compared to the Mediterranean type database to obtain the Mediterranean type, and the supplementary database does not exist but is obtained by the sample test. Type.
• Figure 1 shows the SNP and InDel information analysis process for thalassemia. Detection of copy number variation
• the present invention provides a method for detecting copy number variation, the method comprising detecting a copy number variation portion of the HBB gene and detecting a copy number variation portion of the HBA gene.
• the method includes the following steps:
• the CNV part is divided into the HBB gene detection part and the HBA gene detection part.
• the number of reads of the control gene is calculated; in the HBA gene portion, the number of reads of the HBA 1 gene, the HBA2 gene, and the control gene are detected.
• Detect sample to be read HBB /reads ( control): normal sample reads ( HBB ) /reads ( control ).
• Sample to be tested reads HBAl /reads(control): normal sample reads ( HBAl ) /reads(control);
• Sample to be tested reads HBA2 /reads(control): normal sample reads ( HBA2 ) /reads(control);
• Sample to be tested reads HBAl /reads(HBA2): normal sample reads ( HBAl ) /reads(HBA2).
• the overall variance is ( 19*0.1+69*0.03 ) /88.
• the resulting mean bias and variance are used as fixed errors due to experimental errors, and the theoretical values for each class are regained.
• the theoretical value of the 0.5 category is 0.5-( 0.02*20+0.03*70 ) /90.
• the distance setting can be further away from the class 1 when the distance is limited.
• the distance from 0.8 to 1 is calculated to be 0.2, then the distance from 0.7 to 0.5 is also 0.2, and then 0.15 is set as the limit distance. It is considered that the distance other than 0.15 is not reliable, but if 0.8 is modified to 0.85, then 0.8 belongs to class 1. The distance is 1.5, within the allowable range, and 0.7 does not belong to category 0.5.
• the HBB gene copy number detection information flow and the HBA gene copy number information analysis flow are shown in Fig. 2 and Fig. 3, respectively.
• type of thalassemia refers to the detection of variants of alpha chain and beta chain related genes, including point mutations, insertion deletions and copy number variations, and the like.
• the present invention provides a method for typing thalassemia. Specifically, the following steps are included:
• copy HBB gene copy number variation and data set corresponds to the HBA gene copy number variation and the data set type; at the same time consider the depth of the reading order, mark the label with too few readings; summarize the three parts of the results according to the unified label, get each The complete variation of a sample.
• the main advantages of the invention include:
• the method and system of the present invention have high accuracy, and the data to be tested is relatively low;
• Detection of small fragment insertion and point mutations includes the HBB gene and the HBA gene. After primer-specific capture of genes, different samples were labeled with different indices, and 200-500 bp gel fragments were randomly interrupted and sequenced by high-throughput sequencing. Finally, the reads are divided into different samples and genes using the specificity of index+primer.
• This embodiment uses the BWA comparison software and the samtools software, and the GATK software package as an example.
• BWA comparison software Through the BWA comparison software, these sequences are sequence-aligned with a specific reference sequence, and the comparison result *.sampe file is obtained through two steps of aln and sampe.
• the GATK software package is used to recalibrate the bam file.
• the bwa comparison is not the same for the different reads of the same site.
• the GATK software package is used to recalibrate the low quality SNP near InDel, which is more favorable and accurate. And SNP.
• the original consistency sequence is obtained by the samtools pileup, and the quality value and depth are re-filtered by the program written in the PERL language to obtain the trusted SNP and InDeL.
• the SNP near InDel is corrected, for example, HBB97 C G , which indicates the position of HBB gene 97, and the mutation of ref C is G.
• HBB 97 C -T indicating the position of HBB gene 98, missing a T.
• Such mutations can be replaced by delins, ie insertions after deletion.
• the correction is CT/G, which means that G replaces the CT base of ref.
• SNP and InDel formats are organized, with "gene-location-variation" as the recognition unit, and the type-to-type database is given, and the type of thalassemia is given.
• the variant is not in the database and is marked as "Unknown”.
• the soft chipping position generated in the local coupling algorithm soft chipping is due to the pair end reads in the comparison due to a reads can be Uniq, and the second appears a lot of mismatch, the second reads It will not be discarded, but will be retained and marked s.
• the cleavage sites can be found by using S. These cleavage sites may be caused by large fragment insertion or by deletion. Complex fragments such as insertion, deletion and insertion after deletion can be found by collecting S sites. .
• HBbp gene fragment of 87 bp in length was captured, and each sample was labeled with an index.
• the HBB gene was sequenced using high-throughput sequencing technology.
• the data is indexed to the specificity of the index+primer, and the reads are divided into each sample and each different gene, and the detection is performed simultaneously.
• the recognition sequence is corrected, and the recognition sequence is corrected according to the SNP condition in the same sample due to the mutation of the recognition sequence region.
• an error message is given. Because there are linkages between multiple SNPs, it is not certain that InDel will seriously affect the sequence, so both cases are output.
• the recognition sequence is used to re-divide the reads into each sample to obtain 4 absolute data values, gp: the number of reads of the HBB gene of the sample to be tested, the number of reads of the control sample of the sample to be tested, the number of reads of the normal sample HBB gene, and the normal sample. Control the number of gene reads.
• Ratio sample to be tested HBB gene reads / test sample control gene reads: normal sample HBB gene reads / normal sample control gene reads number.
• HBA1 and HBA2 are very similar in the HBA gene, so only one pair of primers is designed to capture both HBA1 and HBA2.
• the HBA gene fragment of 128 bp in length was captured, and the HBA gene was sequenced by hiseq sequencing technique after index-specific labeling of each sample at both ends. After the machine is down, the data is divided into each sample and each different gene by using the specificity of index+primer, and the detection is performed simultaneously.
• the modified recognition sequence is partially identical to the HBB gene.
• the HBA is divided into HBA1 and HBA2 using the difference sequence of HBA1 and HBA2 from the HBA.
• it is necessary to count 6 original reads, which are normal sample HBA1 gene reads, normal sample HBA2 gene reads, The normal sample control gene reads, the number of HBA1 gene reads in the sample to be tested, the number of HBA2 gene reads in the sample to be tested, and the number of control gene reads in the sample to be tested.
• the HBA part is more complicated, and three ratios need to be calculated, defined as Rl, R2, and R3.
• R 1 sample to be tested HB A 1 gene reads / sample to be tested control gene reads: normal sample HBA 1 gene reads / normal sample control gene reads number.
• test sample control gene reads number normal sample HBA2 gene reads number / normal sample control gene reads number.
• R3 sample to be tested HB A2 gene reads number I sample to be tested HB A 1 gene reads: normal sample HBA2 gene reads / normal sample HBA 1 gene reads number.
• R1 and R2 are used to obtain the absolute value of the HBA1 gene copy number and the absolute value of the HBA2 gene copy number, and finally use R3 for detection.

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