Method for analyzing and predicting alpha globin gene triad by using high-throughput sequencing data

Abstract

The invention relates to a method for predicting an alpha globin gene triad by analyzing high-throughput sequencing data, and belongs to the technical field of gene detection.The method comprises the steps that multiple samples are collected and subjected to high-throughput sequencing, read counts A1 and A2 of non-homologous regions of alpha globin genes HBA1 and HBA2 are obtained, and the A2 / A1 ratio is calculated; the method comprises the following steps: analyzing A2 / A1 ratio data to find that the data is in normal distribution, making a histogram for the A2 / A1 ratio data, determining data in an area greater than u + 2sigma as abnormal data in combination with a normal distribution data distribution rule, and judging that an alpha globin gene triad exists in a sample corresponding to the abnormal data. In the subsequent detection sample inspection, whether the alpha globin gene triad exists in the detection sample can be directly judged according to the A2 / A1 ratio, and compared with the verification of a polymerase chain reaction method, the judgment result of the method is consistent with the result of the polymerase chain reaction method; therefore, the method provided by the invention can be directly used for judging the existence of the alpha globin gene triad.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and in particular, relates to a method for predicting alpha globin gene triplets by using high-throughput sequencing data analysis. Background Art

[0002] Since the hematological indicators of subjects with simple α-globin gene triads are normal, there are rarely detectable clinical phenotypes. However, when α-globin gene triads are combined with β-thalassemia, a variety of clinical phenotypes will appear. The α-globin gene triads will aggravate the imbalance of α-globin chain synthesis, affect the maturation and survival of red blood cells, and play a considerable role in the pathogenesis of thalassemia. Studies have shown that the increase in the number of α-globin gene copies aggravates the imbalance of α and β-globin chains, aggravating the clinical phenotype of the affected subjects and causing mild to severe thalassemia (transfusion-dependent anemia). Therefore, detecting α-globin gene triads can effectively avoid missed diagnosis of thalassemia.

[0003] High-throughput sequencing technology (NGS) has a low error rate and has great advantages in detecting point mutations, but it has some shortcomings in the identification of homologous genes and haplotype analysis. In large population studies using NGS testing, no triplicate or quadruple repeats of the α-globin gene were found, so there are no reports on the detection of α-globin gene triplets using high-throughput sequencing technology. Summary of the invention

[0004] In response to the problems existing in the background technology, the present invention provides a method for predicting α-globin gene triplets by analyzing high-throughput sequencing data, wherein high-throughput sequencing technology is used to sequence the sample target regions HBA1 and HBA2, and by analyzing the intermediate data of NGS, the thalassemia genotype results are accurately identified from the NGS test results, and the α-globin gene triplets are determined, thereby solving the problem of missed diagnosis of thalassemia gene detection caused by the inability of high-throughput sequencing to detect α-globin gene triplets, and providing a new detection method for the detection of α-globin gene triplets.

[0005] To achieve the above object, the present invention is implemented by the following technical solutions:

[0006] The method for predicting α-globin gene triplets by analyzing high-throughput sequencing data is specifically as follows: extracting test sample DNA, performing high-throughput sequencing on the test sample, obtaining target gene fragments of HBA1 and HBA2 of each sample, and obtaining read counts of non-homologous regions of the α-globin gene HBA1 and HBA2 in the test sample, which are respectively recorded as A1 and A2, and calculating the A2 / A1 ratio. When the A2 / A1 ratio of the test sample is greater than 0.8, it is determined that the α-globin gene triplet exists in the test sample; when the A2 / A1 ratio of the test sample is less than 0.8, it is determined that the α-globin gene triplet does not exist in the test sample.

[0007] Furthermore, the read counts of the non-homologous regions of the α-globin gene HBA1 and HBA2 in the intermediate files that passed the quality control were counted and recorded as A1 and A2, respectively. Among them, the target gene single nucleotide polymorphism site SNP minimum sequencing depth greater than 50x is considered to pass the quality control, otherwise it is considered to fail the quality control.

[0008] The method for predicting α-globin gene triplets by using high-throughput sequencing data analysis comprises the following steps:

[0009] (1) 2 ml peripheral blood samples were collected from random individuals, and the sample DNA was extracted. The samples were subjected to high-throughput sequencing to obtain the target gene fragments of HBA1 and HBA2 of each sample. The offline data were preprocessed by the BGI-HALOS data analysis system to obtain the intermediate file of sequencing data;

[0010] (2) Perform quality control analysis on the intermediate files of sequencing data. If the minimum sequencing depth of the single nucleotide polymorphism (SNP) sites of the target gene is greater than 50x, it is considered to be qualified for quality control. Otherwise, it is considered to be unqualified for quality control. Collect the intermediate files that pass the quality control;

[0011] (3) Count the read counts of the non-homologous regions of the α-globin genes HBA1 and HBA2 in the intermediate files that passed the quality control, record them as A1 and A2, respectively, and calculate the A2 / A1 ratio;

[0012] (4) SPSS software was used to make a histogram of the A2 / A1 ratio data to obtain the data mean μ and standard deviation σ. Combined with the distribution law of normal distribution data, abnormal data were identified and the presence of α-globin gene triplets was determined in the abnormal data.

[0013] Furthermore, based on the data mean μ and standard deviation σ obtained from the histogram, it was determined that an α-globin gene triplet existed when the A2 / A1 ratio was greater than u+2σ.

[0014] Furthermore, the obtained data mean μ is 0.6, and the standard deviation σ is 0.1. When the A2 / A1 ratio is greater than u+2σ, it is determined that the α-globin gene triplet exists in the corresponding sample.

[0015] Furthermore, the high-throughput sequencing comprises the following steps:

[0016] S1 Whole-genome DNA extraction

[0017] Peripheral blood samples were collected from the subjects, and genomic DNA of all samples was obtained using a nucleic acid extraction kit. The quality of genomic DNA was measured using a NanoDrop-2000 spectrophotometer, requiring the DNA concentration to be greater than 20 ng / mL and the A260 / A280 ratio to be within the range of 1.8 to 2.0;

[0018] S2, high-throughput sequencing to obtain target gene fragments of HBA1, HBA2, and HBB, including the following steps:

[0019] ① Library construction: The α and β thalassemia gene detection kit was used for library construction. First, the target gene fragments of the samples HBA1, HBA2, and HBB in step S1 were amplified by 4 PCR reaction programs. The PCR amplification products, ≤96 samples, were combined into one library. The DNA sequence ends were repaired, "A" was added to the 3' end, and the adapter sequence was added. Then, DNA fragments were screened. The large-fragment DNA library with a main peak at >500bp was recorded as library A, and the small-fragment DNA library with a main peak at 300bp-500bp was recorded as library B. The library construction was completed;

[0020] ② Library quantification and mixing: Use an ELISA reader or Qubit fluorescence quantification instrument to quantify each library A and each library B. The library with a concentration of ≥3.5ng / μL is qualified, and the library with a concentration below 3.5ng / μL needs to be rebuilt. According to the quantitative results, retain the qualified library with a concentration of ≥3.5ng / μL, mix the qualified libraries with equal quality, and ensure that the total mass of DNA in the mixed library is 168ng. After mixing, use RT2 or TE buffer or nuclease-free water to make up the volume of the mixed library to 48μL, and get the mixed library.

[0021] Hewen Library;

[0022] ③ Target region sequencing, using the universal sequencing reaction kit, combined with the probe anchor sequencing method, using the MGISEQ-2000 sequencer, and using the PE100 sequencing strategy for sequencing. The steps are as follows:

[0023] The library mixed in step ② was subjected to single-stranded DNA, single-stranded DNA cyclization, nucleic acid nanoball preparation, DNB loading, and sequencing; the target genes HBA1, HBA2, and HBB were sequenced to obtain the target gene fragments of HBA1 and HBA2.

[0024] Beneficial effects of the present invention:

[0025] The present invention selects 11,187 random individuals who agree to undergo thalassemia gene testing in the First People's Hospital of Yunnan Province, collects 2 ml of peripheral blood samples for detection and analysis. Extract DNA from all samples respectively, obtain target gene fragments of HBA1, HBA2, and HBB of 11,187 samples, construct a mixed library, perform high-throughput sequencing, obtain read counts A1 and A2 of the non-homologous regions of the α-globin gene HBA1 and HBA2, and calculate the A2 / A1 ratio; by analyzing the A2 / A1 ratio data, it is found that the data is normally distributed, and then the A2 / A1 ratio data is made into a histogram, and combined with the distribution law of normal distribution data, abnormal data is determined, and it is determined that there is an α-globin gene triplet in the sample corresponding to the abnormal data. In the subsequent test sample inspection, the abnormal value can be directly referred to, and the presence of α-globin gene triplet in the test sample can be determined based on the A2 / A1 ratio. The determination result of the present invention has been verified and compared with the polymerase chain reaction method, and the results of the two are consistent, indicating that the method of the present invention can be directly used to determine the presence of α-globin gene triplet. The present invention improves the determination efficiency of α-globin gene triplet.

[0026] The present invention uses high-throughput sequencing technology to sequence samples, and through analysis of intermediate data of NGS, accurately identifies the thalassemia genotype results from the NGS test results, determines the α-globin gene triplet, and solves the problem of missed diagnosis of thalassemia gene detection caused by the inability of high-throughput sequencing to detect the α-globin gene triplet, and provides a new detection method for the detection of the α-globin gene triplet. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a PP diagram of Example 1 of the present invention;

[0028] Figure 2 It is a 68-95-99.7 rule diagram of the normal distribution data distribution law of Example 1 of the present invention;

[0029] Figure 3 This is a statistical analysis chart of the ratio of A2 / A1 of the non-homologous position sequence numbers of α-globin genes HBA1 and HBA2 in Example 1 of the present invention;

[0030] Figure 4 This is the sample electrophoresis diagram of Example 2 of the present invention. DETAILED DESCRIPTION

[0031] In order to make the purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention will be described in detail below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0032] In order to more clearly illustrate the present invention, it is described in detail through the following examples.

[0033] Example 1

[0034] The method for predicting alpha globin gene triplets by using high-throughput sequencing data analysis comprises the following steps:

[0035] (1) Whole genome DNA extraction

[0036] 11187 random individuals who were informed and consented to thalassemia gene testing during prenatal eugenics examination or prenatal screening at Yunnan Provincial First People's Hospital were selected to collect 2 ml peripheral blood samples. Genomic DNA of all samples was obtained using a nucleic acid extraction kit produced by BGI Biotech (Wuhan) Co., Ltd., and the quality of genomic DNA was measured using a NanoDrop-2000 spectrophotometer, requiring the DNA concentration to be greater than 20 ng / mL and the A260 / A280 ratio to be in the range of 1.8 to 2.0.

[0037] (2) High-throughput sequencing was used to obtain the target gene fragments of HBA1, HBA2, and HBB.

[0038] ①Library construction

[0039] The library was constructed using the α and β thalassemia gene detection kit produced by BGI Biotech (Wuhan) Co., Ltd. First, the target gene fragments of the samples HBA1, HBA2, and HBB in step (1) were amplified by four PCR reaction programs, and the PCR amplification products (≤96 samples) were merged into a library. The DNA sequence ends were repaired, "A" was added to the 3' end, and the adapter sequence was added. Then, the DNA fragments were screened. The large-fragment DNA library with a main peak at >500bp was library A, and the small-fragment DNA library with a main peak at 300bp-500bp was library B. The library construction was completed. The specific operation steps are shown in the kit instructions.

[0040] ②Library quantification and mixing

[0041] Use an ELISA reader or Qubit fluorescence quantifier to quantify each library A and each library B. The library with a concentration of ≥3.5ng / μL is qualified, and the library with a concentration below 3.5ng / μL needs to be rebuilt. According to the quantitative results, the qualified library with a concentration of ≥3.5ng / μL is retained, and the qualified libraries are mixed with equal quality to ensure that the total mass of DNA in the mixed library is 168ng. After mixing, use RT2 or TE buffer or nuclease-free water to make up the volume of the mixed library to 48μL to obtain a mixed library.

[0042] ③ Target region sequencing was performed using a universal sequencing reaction kit (combined probe anchor sequencing method) produced by Wuhan MGI Intelligent Manufacturing Technology Co., Ltd. and a MGISEQ-2000 sequencer (MGI, Wuhan, China) using the PE100 sequencing strategy. The steps are as follows:

[0043] The library mixed in step ② was subjected to DNA single-stranding, single-stranded DNA cyclization, nucleic acid nanoball preparation, DNB loading, and sequencing. The specific operation steps are shown in the kit instructions. The target genes (HBA1, HBA2, HBB) were sequenced.

[0044] (3) Sequencing data preprocessing

[0045] The sequencing data was preprocessed by the BGI-HALOS data analysis system to obtain the sequencing data intermediate file.

[0046] (4) Perform α-globin gene triplet prediction analysis on the sequencing intermediate file obtained in step (3)

[0047] ①Analyze the sample quality control data (sample qc) in the intermediate file for quality control analysis. First, analyze the sequencing depth of the HBA1, HBA2, and HBB target genes of each sequencing sample. The minimum sequencing depth of the single nucleotide polymorphism sites (SNPs) of the three target genes must be greater than 50x, that is, HBA1_Minimum_depth>50x, HBA2_Minimum_depth>50x, HBB1_Minimum_depth>50x. If this condition is met, the quality control passes, otherwise it fails. According to the quality control results, remove the sample data that failed the quality control and obtain the intermediate file that passed the quality control.

[0048] ② Further analyze the data of all samples that passed quality control, and count the read counts (reads) of the non-homologous regions of the α-globin gene HBA1 and HBA2 in the intermediate file (Map_Stat) data that passed quality control as A1 and A2, respectively, and calculate the A2 / A1 ratio.

[0049] ③ Use SPSS software to analyze the A2 / A1 ratio data and obtain the normal PP diagram of the A2 / A1 ratio (see Appendix Figure 1 ), we can see from the figure that the data is distributed near the straight line, the data is normally distributed, and can be processed according to the rules of normally distributed data.

[0050] ④ Use SPSS software to make a histogram of the A2 / A1 ratio data (attached Figure 3 ), and get the data average μ

[0051] =0.6, standard deviation σ =0.1. Combined with the normal distribution data distribution range theoretical model (Appendix Figure 2 )Analyze the A2 / A1 ratio data.

[0052] Combined with the normal distribution data distribution law 68-95-99.7 rule diagram ( Figure 2 ) indicates the probability of data falling within a specific standard deviation (σ) in a normal distribution. 68.27% (about 68%) of the data fall within the range of mean (μ) ± 1 standard deviation (σ), which is represented by the pink area in the figure, 95.45% (about 95%) of the data fall within the range of mean (μ) ± 2 standard deviations (σ), which is represented by the pink and purple areas in the figure, and 99.73% (about 99.7%) of the data fall within the range of mean (μ) ± 3 standard deviations (σ), which is represented by the pink, purple and green areas in the figure. The histogram of the A2 / A1 ratio data found that 96.5% of the values ​​are distributed in the range of 0.40 to 0.80, which is consistent with the distribution law of normal distribution data [u-2σ, u+2σ], and the data with an A2 / A1 ratio greater than u+2σ is determined to be abnormal.

[0053] ⑤ Samples in the interval where the A2 / A1 ratio is greater than u+2σ(0.80) predict the occurrence of α-globin gene duplication, that is, the presence of α-globin gene triplets.

[0054] ⑥ Determine the test samples with A2 / A1 ratio ≥ 0.80 and judge that they have α-globin gene triplet.

[0055] Example 2 Results Verification

[0056] Random individuals who underwent thalassemia gene testing in the First People's Hospital of Yunnan Province were selected to collect 2 ml peripheral blood samples for testing. Here we randomly selected three test samples (numbered 23800012588-1, 23800012589-1, and 23800012629-1) for specific analysis and result verification as follows:

[0057] (1) Follow steps (1) to (3) in Example 1 to obtain the intermediate file of sequencing data of each test sample.

[0058] (2) Using the same method as step (4) of Example 1, remove the sample data that failed the quality control to obtain the intermediate file that passed the quality control;

[0059] (3) The intermediate files that passed the quality control were further analyzed. The read counts of the non-homologous regions of the α-globin genes HBA1 and HBA2 in the data of the intermediate files that passed the quality control (Map_Stat) were recorded as A1 and A2 respectively, and the A2 / A1 ratio was calculated. The results are shown in the following table.

[0060] Test sample number A1 counting results A2 counting results A2 / A1 23800012588-1 3476 2148 0.62 23800012589-1 328 356 1.09 23800012629-1 6197 5596 0.90

[0061] The A2 / A1 ratios of the three test samples 23800012588-1, 23800012589-1, and 23800012629-1 were 0.62, 1.09, and 0.90, respectively. It was judged that the α-globin gene triplet was not present in the test sample 23800012588-1, and the α-globin gene triplet was present in the test samples 23800012589-1 and 23800012629-1.

[0062] Verification results:

[0063] The polymerase chain reaction (PCR) method was used to verify the test results of the three test samples 23800012588-1, 23800012589-1, and 23800012629-1. The verification method is as follows:

[0064] The DNA of the three test samples were amplified by PCR respectively. The amplification reaction system and reaction conditions were as follows, and the amplified products were analyzed by electrophoresis.

[0065] PCR reaction system:

[0066] Reagent name Amount (μL) / reaction ApexHF Hs CL buffer 7.5 ApexHF Hs DNA polymerase 0.2 Cresol red (1 mg / mL) 1 Triplet Primer 0.5 ddH2O 3.8 Template DNA 2

[0067] PCR reaction conditions:

[0068]

[0069]

[0070] Agarose gel electrophoresis analysis of PCR amplification products

[0071] Agarose gel electrophoresis conditions: voltage 150V, current 400mA, time 80min. According to the analysis results of the agarose gel imager, a band of about 2000bp appeared and was interpreted as ααα anti3.7 / αα, a band of about 1800 bp appeared and was interpreted as ααα anti4.2 / αα, the internal reference gene fragment band size is about 2200bp. If only the internal reference fragment size band appears, it will be interpreted as αα / αα.

[0072] The agarose gel imager images of the three test samples of this Example 2 are shown in Figure 4 ( Figure 4In the analysis, 12589 represents sample 23800012589-1, 12588 represents sample 23800012588-1, and 12629 represents sample 23800012629-1). According to the analysis results of the agarose gel imager, sample 23800012588-1 was interpreted as αα / αα, and there was no α-globin gene triplet; samples 23800012589-1 and 23800012629-1 were interpreted as ααα anti4.2 / αα and ααα anti3.7 / αα, there is an α-globin gene triplet, and the polymerase chain reaction (PCR) result is consistent with the predicted result of the present invention.

[0073] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A method for predicting α-globin gene triplets using high-throughput sequencing data analysis, characterized in that: The DNA of the test samples was extracted, and high-throughput sequencing was performed on the test samples to obtain the target gene fragments of HBA1 and HBA2 of each sample, and the read counts of the non-homologous regions of the α-globin genes HBA1 and HBA2 in the test samples were obtained, which were recorded as A1 and A2, respectively. The A2 / A1 ratio was calculated. When the A2 / A1 ratio of the test sample was greater than 0.8, it was determined that the α-globin gene triplet existed in the test sample; when the A2 / A1 ratio of the test sample was less than 0.8, it was determined that the α-globin gene triplet was not present in the test sample.

2. A method for predicting α-globin gene triplets using high-throughput sequencing data analysis, characterized in that: The following steps are involved: (1) 2 ml peripheral blood samples were collected from random individuals, and the sample DNA was extracted. The target gene fragments of HBA1 and HBA2 of each sample were obtained by high-throughput sequencing. The offline data were pre-processed by the BGI-HALOS data analysis system to obtain the intermediate file of sequencing data; (2) Perform quality control analysis on the intermediate files of sequencing data. If the minimum sequencing depth of the single nucleotide polymorphism (SNP) sites of the target gene is greater than 50x, it is considered to be qualified for quality control. Otherwise, it is considered to be unqualified for quality control. Collect the intermediate files that pass the quality control. (3) Count the read counts of the non-homologous regions of the α-globin genes HBA1 and HBA2 in the intermediate files that passed quality control, record them as A1 and A2, respectively, and calculate the A2 / A1 ratio; (4) SPSS software was used to make a histogram of the A2 / A1 ratio data to obtain the data mean μ and standard deviation σ. Combined with the distribution law of normal distribution data, abnormal data were identified and the presence of α-globin gene triplets was determined in the abnormal data.

3. The method for predicting α-globin gene triplets using high-throughput sequencing data analysis according to claim 2, characterized in that: Based on the data mean μ and standard deviation σ obtained from the histogram, it is determined that the α-globin gene triplet exists when the A2 / A1 ratio is greater than u+2σ.

4. The method for predicting α-globin gene triplets using high-throughput sequencing data analysis according to claim 3, characterized in that: The average value μ of the data obtained is 0.6, and the standard deviation σ is 0.

1. When the A2 / A1 ratio is greater than u+2σ, the α-globin gene triplet exists in the corresponding sample.

5. The method for predicting α-globin gene triplets using high-throughput sequencing data analysis according to claim 1 or 2, characterized in that: The high-throughput sequencing comprises the following steps: S1 Whole genome DNA extraction Peripheral blood samples were collected from the subjects, and genomic DNA of all samples was obtained using a nucleic acid extraction kit. The quality of genomic DNA was measured using a NanoDrop-2000 spectrophotometer, requiring the DNA concentration to be greater than 20 ng / mL and the A260 / A280 ratio to be within the range of 1.8 to 2.0; S2, high-throughput sequencing to obtain target gene fragments of HBA1, HBA2, and HBB, including the following steps: ① Library construction: The α and β thalassemia gene detection kit was used for library construction. First, the target gene fragments of the samples HBA1, HBA2, and HBB in step S1 were amplified by 4 PCR reaction programs. The PCR amplification products, ≤96 samples, were combined into one library. The DNA sequence ends were repaired, "A" was added to the 3' end, and the adapter sequence was added. Then, DNA fragments were screened. The large-fragment DNA library with a main peak at >500bp was recorded as library A, and the small-fragment DNA library with a main peak at 300bp-500bp was recorded as library B. The library construction was completed; ② Library quantification and mixing: Use an ELISA reader or Qubit fluorescence quantification instrument to quantify each library A and each library B. The library with a concentration ≥3.5ng / μL is qualified, and the library with a concentration below 3.5ng / μL needs to be rebuilt. According to the quantitative results, retain the qualified library with a concentration ≥3.5ng / μL, mix the qualified libraries with equal quality, and ensure that the total mass of DNA in the mixed library is 168ng. After mixing, use RT2 or TE buffer or nuclease-free water to make up the volume of the mixed library to 48μL to obtain a mixed library; ③ Target region sequencing, using the universal sequencing reaction kit, combined with the probe anchor sequencing method, using the MGISEQ-2000 sequencer, and using the PE100 sequencing strategy for sequencing. The steps are as follows: The library mixed in step ② was subjected to single-stranded DNA, single-stranded DNA cyclization, nucleic acid nanoball preparation, DNB loading, and sequencing; the target genes HBA1, HBA2, and HBB were sequenced to obtain the target gene fragments of HBA1 and HBA2.

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