CN111235239A - Multiple PCR _ SNP genotyping detection method - Google Patents

Abstract

The invention belongs to the technical field of genotyping detection, and discloses a multiplex PCR_SNP genotyping detection method. The multiplex PCR_SNP genotyping detection method includes: primer design; DNA extraction from tissue, cells and blood samples; PCR amplification; PCR product Purification; single base extension; resin purification: each extension reaction product was purified with 6 mg of Clean Resin resin; chip spotting and mass spectrometry detection. The multiplex PCR_SNP genotyping detection method provided by the present invention shows that among the 65 typing sites, there are 6 sites with no information and a typing success rate of less than 95%, accounting for 9.23%; the MAF is relatively low SNP sites There were 6 SNP sites, accounting for 9.23%; HWE test p<1e‑2 SNP sites were 7, accounting for 10.77%; the final effective SNP sites used were 59, accounting for 90.77%.

Description

A kind of multiplex PCR_SNP genotyping detection method

technical field

The invention belongs to the technical field of genotyping detection, and in particular relates to a multiplex PCR_SNP genotyping detection method.

Background technique

At present, the closest prior art: polymerase chain reaction (Polymerase Chain Reaction, PCR) is based on the premise that the template single strand and the primer strand are correctly paired according to the base pairing principle. Therefore, in the area containing base differences, design a set of PCR primers with only a single base difference, use a common PCR amplifier to amplify, and detect whether an amplification product that meets the designed fragment length is produced, and the target can be judged. The base type of the target site of the region. However, when using conventional PCR primers, even if there is a single base mismatch, the PCR reaction can sometimes proceed normally, and an amplification product with the same length as the target fragment can be obtained. Therefore, when using conventional primers for PCR amplification typing, false positive results cannot be excluded, and wrong genotyping results may be obtained.

At present, the commonly used methods for genotype detection of SNP loci mainly include sequencing method, chip method and mass spectrometry method, among which sequencing method is relatively expensive, while chip method and mass spectrometry method are only used for simultaneous determination of multiple loci or when studying large groups of samples. It has many advantages, and all three methods must be supported by the relevant large-scale instrument platform, and there are many limitations. Therefore, a new multiplex PCR_SNP genotyping detection method is urgently needed to make up for the deficiencies of the prior art.

To sum up, the problems existing in the prior art are: among the existing SNP locus genotype detection methods, the sequencing method is relatively expensive, while the chip method and mass spectrometry method can only be used to simultaneously measure multiple loci or study large population samples. It has many advantages, and all three methods must be supported by the relevant large-scale instrument platform, and there are many limitations.

SUMMARY OF THE INVENTION

Aiming at the problems existing in the prior art, the present invention provides a multiplex PCR_SNP genotyping detection method.

The present invention is achieved in this way, a multiple PCR_SNP genotyping detection method, when the multiple PCR_SNP genotyping detection method PCR amplification, when the 3'-end base of the primer is completely matched with the template, the DNA polymerase carries out an extension reaction , the detection signal is strong; if the bases at the 3' end of the primer do not match the template, the detection signal is weak; design different primers according to the genotype of the allele to distinguish different alleles. The 5 ends of the primers of the allele are respectively connected with a different tag sequence, and the sequence can hybridize with the tag sequence ordered on the chip to realize the interpretation of the genotype.

Further, the four SNP sites of the multiplex PCR_SNP genotyping detection method design primers, and connect Tag1/Tag2, Tag3/Tag4, Tag5/Tag6, Tag7/Tag8 at the 5' ends of the upstream primers, and simultaneously use these four For Tag spotting, 3 spots were repeated for each Tag, and 3 hybridization positive quality control spots PC, 3 hybridization negative quality control spots NC, 3 Blank spots, and 3 Hex spots were spotted at the same time.

Further, the multiplex PCR_SNP genotyping detection method includes designing PCR amplification primers and single-base extension primers for the SNP sites to be detected.

Further, the multiplex PCR_SNP genotyping detection method includes extracting DNA in the blood sample, quantifying with a spectrophotometer, performing quality inspection on agarose gel electrophoresis, adjusting the concentration of qualified DNA to 50ηg/ul, and storing at -20°C for later use. .

Further, the multiplex PCR_SNP genotyping detection method comprises PCR amplification, using multiplex PCR amplification technology, the total volume of each reaction is 5ul, including template DNA 10ng, Hotstar Taq 0.5U, each amplification primer 0.5pmol, 25mM dNTPs , 0.1ul, the reaction conditions are 94 ℃ for 4 minutes; 94 ℃ for 20 seconds, 56 ℃ for 30 seconds, 72 ℃ for 1 minute, 45 cycles; 72 ℃ for 3 minutes; 4 ℃.

Further, the multiplex PCR_SNP genotyping detection method includes the purification of PCR products, the PCR reaction products are treated with 0.5U SAP, the free dNTPs in the system are removed, and the reaction system is 7 μl, wherein the PCR product is 5 μl, the SAP mixture is 2 μl, and the SAP is 0.5U, buffer 0.17μl, the reaction program is 37°C for 20 minutes; 85°C for 5 minutes; 4°C.

Further, the multiplex PCR_SNP genotyping detection method includes single base extension, the total volume of 9 μl reaction system contains 7 μl of PCR product after SAP treatment, wherein each extension reaction primer mixture is 0.804l, iPLEX enzyme is 0.041l, and extension mixture is 0.2μl. The reaction program was 94°C for 30 seconds; 94°C for 5 seconds; 52°C for 5 seconds, 80°C for 5 seconds for 5 cycles; return to 94°C for 5 seconds, a total of 40 cycles; 72°C for 3 minutes, 4°C.

Further, the multiplex PCR_SNP genotyping detection method comprises resin purification, and each extension reaction product is purified with 6mg Clean Resin resin;

After chip spotting and mass spectrometry detection, the purified product was transferred to a 384-well SpectroCHIP chip and measured on the machine. SpectroCHIP chip uses MALDI-TOF matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis, and the detection results are typed and output by software.

Further, the multiplex PCR_SNP genotyping detection method includes

(1) Data preprocessing, filtering out non-informative sites in all samples and sites with typing success rate less than 90%; filtering out sites with MAF less than 0.01 in control samples; using control samples, for each Hardy-Weinberg equilibrium test for each SNP site;

(2) Single-point association analysis method, for each locus after preprocessing, the chi-square test and Fisher's exact test were used to test the differences between the cases and controls for the four cases, and for the cases where the odds ratio could be calculated, calculate Odds ratios and their 95% confidence intervals.

Further, the multiplex PCR_SNP genotyping detection method includes four situations:

(1) Perform chi-square test and Fisher exact test on allele frequencies and calculate OR value and 95% confidence interval;

(2) Chi-square test and Fisher's exact test for genotype frequencies;

(3) Chi-square test and Fisher's exact test were performed on the genotype frequencies for the dominant model, and the OR value and 95% confidence interval were calculated;

(4) Chi-square test and Fisher's exact test were performed on the genotype frequencies for the recessive model, and the OR value and 95% confidence interval were calculated.

To sum up, the advantages and positive effects of the present invention are as follows: in the multiplex PCR_SNP genotyping detection method provided by the present invention, experiments show that in the 65 typing loci, there is no information amount and typing success rate (call rate). There are 6 sites with less than 95% (9.23%), 6 SNP sites with low MAF (9.23%), and 7 SNP sites with p<1e-2 by HWE test (10.77%). The effective SNP site used was 59 (90.77%). Association analysis was performed for each locus after preprocessing. Four significant loci with a Fisher's exact test p value less than 0.05 were selected, and combined with the Hardy-Weinberg balance test results and typing quality of these loci to give a comprehensive score, the highest score was 5, and the lowest score was 5. is 1.

Description of drawings

FIG. 1 is a flowchart of a multiplex PCR_SNP genotyping detection method provided in an embodiment of the present invention.

FIG. 2 is a schematic diagram of the multiplex PCR_SNP genotyping detection principle provided in Example 1 of the present invention.

FIG. 3 is a diagram of the experimental design of the multiplex PCR_SNP genotyping detection provided in Example 1 of the present invention.

4-6 are graphs of the results of the multiplex PCR_SNP genotyping detection experiment provided in Example 1 of the present invention.

7 is a flow chart of the PCR-SNP sequencing method provided in Example 2 of the present invention.

8 is a schematic diagram of the quality control results of agarose gel electrophoresis provided in Example 2 of the present invention (scheme A-90C).

9 is a schematic diagram of the quality control results of agarose gel electrophoresis provided in Example 2 of the present invention (scheme B-3C).

Figure 10 is a schematic diagram of the quality control results of fluorescence quantitative PCR provided in Example 2 of the present invention (scheme A-90C).

Figure 11 is a schematic diagram of the quality control results of fluorescence quantitative PCR provided in Example 2 of the present invention (scheme B-3C).

Detailed ways

In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, but not to limit the present invention.

In view of the problems existing in the prior art, the present invention provides a multiplex PCR_SNP genotyping detection method. The present invention will be described in detail below with reference to the accompanying drawings.

As shown in Figure 1, the multiplex PCR_SNP genotyping detection method provided in the embodiment of the present invention comprises the following steps:

S101: Primer design.

S102: DNA extraction from tissues, cells and blood samples.

S103: PCR amplification.

S104: PCR product purification.

S105: Single base extension.

S106: Resin purification: Each extension reaction product was purified with 6 mg of Clean Resin resin.

S107: Chip spotting and mass spectrometry detection.

The technical solutions of the present invention will be further described below in conjunction with the embodiments.

Embodiment 1: Multiplex PCR_SNP genotyping detection experiment

1. Materials

Blood samples were taken from 130 pregnant women, including 79 normal patients and 51 diabetic patients. 2ml of whole blood was prepared for each sample. Immediately after the blood was drawn, an EDTA anticoagulation tube (BD Company, USA) was added, and it was immediately inverted and mixed. save in.

2. Method

2.1 The principle of chip experiment

As shown in Figure 2, the principle of general chip technology is: during PCR amplification, the DNA polymerase can effectively carry out the extension reaction when the 3'-end base of the primer is completely matched with the template, and the final detection signal is strong; If the base at the 3' end does not match the template, the extension efficiency will be greatly reduced, and the final detection signal will be weak. Different alleles can be distinguished by designing different primers according to the different genotypes of the alleles. A different tag sequence is connected to the 5 ends of the primers of different alleles, and these sequences can hybridize with the tag sequences spotted on the chip, so as to realize the interpretation of the genotype.

2.2 Experimental Design

As shown in Figure 3, primers were designed for the four SNP sites selected in the previous experiment, and Tag1/Tag2, Tag3/Tag4, Tag5/Tag6, and Tag7/Tag8 were connected to the 5' ends of the upstream primers. For Tag spotting chip, 3 spots are repeated for each Tag, and 3 hybridization positive quality control spots (PC), 3 hybridization negative quality control spots (NC), and Blank spots (1* spotting solution) are spotted at the same time. 3, Hex points 3.

2.3 Chip experiment steps

分子量阵列技术(美国Sequenom公司)，对130例样本的65个SNP位点进行分型，具体如下：Screened 70 SNP sites reported in literature and related to diabetes, after preliminary primer design, 65 of them were selected (Appendix 2), using Sequenom

Molecular weight array technology (Sequenom, USA) was used to type 65 SNP loci in 130 samples, as follows:

2.3.1 Primer Design

Genotyping Tools and MassARRAY Assay Design software of Sequenom Company were used to design PCR amplification primers and single-base extension primers of the SNP site to be tested, and the primers were synthesized by Beijing Synthesis Department of Innitrogen Company.

2.3.2 DNA extraction from tissues, cells and blood samples

DNA in blood samples was extracted using Wizard Genomic DNA purification Kit (Promega), quantified by spectrophotometer, and checked by agarose gel electrophoresis. The DNA that passed the quality inspection was adjusted to a concentration of 50ηg/ul and stored at -20°C for later use.

2.3.3 PCR amplification

Using multiplex PCR amplification technology, the total volume of each reaction is 5ul, including template DNA 10ηg, Hotstar Taq 0.5U, each amplification primer 0.5pmol, 25mM dNTP, 0.1ul, the reaction conditions are 94 °C for 4 minutes; 94 °C 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, 45 cycles; 72°C for 3 minutes; 4°C.

2.3.4 PCR product purification

The PCR reaction product was treated with 0.5U SAP (shrimp alkaline phosphatase) to remove free dNTPs in the system. The reaction system was 7 μl, including 5 μl of PCR product and 2 μl of SAP mixture (SAP 0.5U, buffer 0.17 μl). Reaction program 37°C for 20 minutes; 85°C for 5 minutes; 4°C.

2.3.5 Single base extension

The total volume of the 9 μl reaction system contained 7 μl of the PCR product after SAP treatment, wherein each extension reaction primer mix was 0.804 l, iPLEX enzyme was 0.041 l, and the extension mixture was 0.2 μl. The reaction program was 94°C for 30 seconds; 94°C for 5 seconds; 52°C for 5 seconds, 80°C for 5 seconds for 5 cycles; return to 94°C for 5 seconds, a total of 40 cycles; 72°C for 3 minutes, 4°C.

2.3.6 Resin purification

Each extension reaction product was purified with 6 mg of Clean Resin resin.

2.3.7 Chip spotting and mass spectrometry detection

The purified product was transferred to a 384-well SpectroCHIP (Sequenom) chip and measured on the machine. SpectroCHIP chip uses MALDI-TOF (matrix-assisted laser desorption/ionization-time offlight) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis, and the detection results are typed and output using TYPER 4.0 software (sequenom).

3. Correlation analysis method

3.1 Data preprocessing

(1) Filter out sites with no information in all samples and sites with a call rate less than 90%;

(2) filter out the sites with MAF (minor allele frequency) less than 0.01 in the control sample;

(3) Using the control sample, carry out Hardy-Weinberg Equilibrium (HWE) test for each SNP site.

3.2 Single point correlation analysis method

For each locus after preprocessing, chi-square test and Fisher's exact test were used to test the differences between cases and controls for the four cases, and for the cases where the odds ratio (OR) could be calculated, calculate the odds ratio and Its 95% confidence interval.

The four cases are as follows:

1. Perform chi-square test and Fisher exact test on allele frequencies and calculate OR value and 95% confidence interval. The specific data form is shown in Table 1.

Table 1 Allele frequencies for experimental data

Note: B is used as a reference when calculating the OR value

2. Chi-square test and Fisher's exact test were performed on the genotype frequencies, and the specific data forms are shown in Table 2.

Table 2 Genotype frequencies for experimental data

3. Perform chi-square test and Fisher's exact test on the genotype frequencies for the dominant model and calculate the OR value and 95% confidence interval. The specific data form is shown in Table 3.

Table 3. Experimental data on genotype frequency by dominant model

Note: BB is used as a reference when calculating the OR value

Among them, AA is the wild homozygous genotype.

4. Perform chi-square test and Fisher's exact test on the genotype frequencies for the recessive model and calculate the OR value and 95% confidence interval. The specific data form is shown in Table 4.

Table 4. Experimental data of recessive model on genotype frequency

Note: BB+AB is the reference when calculating the OR value

Among them, AA is the wild homozygous genotype.

5. Results

Among the 65 typing loci, there are 6 loci (9.23%) with no information and a call rate of less than 95%, and 6 SNP loci with low MAF (9.23%) , HWE test p<1e-2 SNP site is 7 (accounting for 10.77%), and the final effective SNP site used is 59 (accounting for 90.77%).

Association analysis was performed for each locus after preprocessing. Four significant loci with a Fisher's exact test p value less than 0.05 in the above four cases were selected, and combined with the Hardy-Weinberg equilibrium test results and typing quality of these loci to give a comprehensive score, the highest It is divided into 5 and the lowest score is 1. The 4 significant loci are shown in Table 5.

Table 5 4 significant loci

(Note: SNPname: SNP number, HWE-pValue: HWE-pValue, SNP Call Rate: SNP callrate value, Score: Comprehensive score).

As shown in Figure 4, the quality of the chip can be checked by hybridizing the artificially synthesized cTag complementary to the Tag sequence with the chip. In this experiment, the cTag sequences of Tag1, 2, 3, 4, 7, and 8 and the complementary sequences of PC were used to hybridize with the chip. It can be seen that the signals of each Tag point, PC point and HEX point are relatively strong, while NC point and BC point have strong signals. Then there is no signal, cTag5 and cTag6 are not added, and there is no signal at the corresponding site, which proves that the quality of the chip is no problem.

As shown in Figure 5, the primers of the four sites were individually amplified by PCR, and the size of the product bands displayed by electrophoresis was consistent with the expected. It is shown that the primer design and synthesis are no problem.

As shown in Figure 6, two samples from diabetic individuals and two samples from normal individuals were taken and hybridized with the chip after quadruple PCR amplification. Compared with the experimental results of Sequenom, the position of the red box is the expected strong positive point, and the blue box The position is the expected negative point with relatively weak signal, and the white box is the quality control point. From the experimental results, among the four sites, the expected signals of positive sites are all better, and the signals of positive sites of three SNP sites are higher than those of negative sites. But the signal of site 2 negative point is as strong as that of positive point. Three of the four loci performed well, and the genotype could be judged by the ratio of the signal values of the positive and negative loci.

Example 2: PCR-SNP sequencing experiment

1. Project process

The main process of the project is shown in Figure 7. The target region resequencing combines multiple PCR technology and high-throughput sequencing technology to design specific primers for the sites to be detected, and perform multiple PCR amplification in a single tube. Barcode primers differentiate and perform high-throughput sequencing of the amplicons. The sequencing results use bioinformatics methods to distinguish different samples and finally obtain mutation information for each locus.

2. Materials and methods

2.1 Experimental materials

2.1.1 Experimental instrument

The main experimental instruments and manufacturers are shown in Table 6.

Table 6 Main experimental instruments and manufacturers

2.1.2 Experimental reagents

The main experimental reagents, consumables and manufacturers are shown in Table 7.

Table 7 Main experimental reagents, consumables and manufacturers

2.1.3 Software and Network Resources

The main experimental software and network resources are shown in Table 8.

Table 8 Main experimental software and network resources

2.2 Experimental method

2.2.1 Target gene primer sequences

2.2.2 Library preparation

2.2.2.1 Primer adjustment for multiplex PCR

1. One round of PCR

The first round of amplification was performed on the quality control product and Nbutin C with the multiple primer working solution. The amplification system and reaction conditions are shown in Table 9, and the PCR program is shown in Table 10.

Table 9 Amplification system

Table 10 PCR program

2. Two rounds of PCR

Add 20100u1 dd day to each well of one round product, centrifuge briefly, and let stand for 10min at room temperature. The diluent was used as the second round of amplification template, the amplification system and reaction conditions were shown in Table 11, and the PCR program was shown in Table 12.

Table 11 Amplification system

Table 12 PCR program

3. After the amplification, the PCR product was subjected to electrophoresis detection, 3% agarose gel, and the PCR product was loaded with 5u1 to observe whether the electrophoresis bands were uniform and whether there were any miscellaneous bands.

4. Determine the number of diluted aliquots according to the multiplex of multiplex PCR (detection limit of 384 plates at a time). A small sample was diluted to ^10-4 for qPCR detection. After the experiment, adjust the initial multiplex primer mix according to the Ct value. The qPCR amplification system is shown in Table 13, and the qPCR program is shown in Table 14.

Table 13 qPCR amplification system

Table 14 qPCR program

2.2.2.2 Product production

1. One round of PCR

Take the adjusted polyplex working solution to prepare PCR mix, and distribute it in 96-well plates corresponding to the required number. Take the sample plate and fully thaw it, shake it, centrifuge it at 1000rpm, and then add the sample. When adding samples, 2 wells were reserved in each plate, and positive and negative controls were added respectively. The one-round amplification system is shown in Table 15, and the PCR program is shown in Table 16.

Table 15 PCR amplification system

Table 16 PCR program

2. Two rounds of PCR

Add 100u1 of ddH ₂ O to each well of one round of products, centrifuge briefly, and let stand at room temperature for 10 min. The diluent was used as the template for the second round of amplification, the amplification system was shown in Table 17, and the PCR program was shown in Table 18.

Table 17 PCR amplification system

Table 18 PCR program

3. After the amplification, the PCR product is subjected to electrophoresis detection, 3% agarose gel, and 5 ul of the PCR product is loaded to observe whether the electrophoresis bands are uniform and whether there are any miscellaneous bands.

2.2.2.3 Library Mixing

1. Take one 96-well plate. Adjust the gun to 5ul, insert the Axygen 10ul pipette tip, and transfer 5ul of the product to each well of the PCR plate and transfer it to the U-shaped groove.

2. Take 1 round-bottom centrifuge tube, unscrew the cap, and wrap the spiral mouth of the round-bottom centrifuge tube with parafilm for 2 circles for later use. Note that the winding is clockwise.

3. Take a 200ul pipette, insert the Axygen tip, and transfer the product mix in the U-shaped groove to a round-bottomed centrifuge tube.

4. Tighten the cap and vortex for 30 seconds.

5. Fix the centrifuge tube on the shaker in parallel, start the shaker, observe the movement of the liquid in the tube, adjust the shaker to a suitable speed, and the maximum liquid amplitude in the tube is appropriate, and shake the product mix overnight.

2.2.2.4 Purification

1. Load the mixed library into electrophoresis. After electrophoresis, cut out the target fragments, chop the fragments, divide them into 4 parts and place them in 4 1.5ml low adsorption EP tubes. The printed tube containing the gel was weighed, the tube weight was subtracted, and the gel mass was calculated and recorded on the tube cap. Add an equal volume of solution PN to the gel block (if the gel weight is 0.1g, and its volume can be regarded as 100ul, then add 100ul of PN solution), place it in a water bath at 50°C, and gently turn the centrifuge tube up and down to ensure the gel block. Fully dissolved. If there are still undissolved glue pieces, you can continue to stand for a few minutes or add some more sol solution until the glue pieces are completely dissolved.

2. When the gel dissolves, put 4 adsorption columns into the collection tube, add 500ul of equilibrium solution BL to the adsorption column, centrifuge at 12,000 rpm for 1 min, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube .

3. Dissolve the melted gel to room temperature, add 4 adsorption columns CA2 (the adsorption columns are placed in the collection tube), leave at room temperature for 2 min, centrifuge at 12,000 rpm for 30 s, discard the waste liquid, and put the adsorption column CA2 into the collection tube in the tube. (The volume of the adsorption column is 800ul, if the sample volume is greater than 800ul, it can be added in batches)

4. Add 600ul of rinsing solution PW to the adsorption column CA2 (please check whether absolute ethanol has been added before use), let it stand for 2 minutes, centrifuge at 12000rpm for 30sec, pour out the waste liquid in the collection tube, and put it into the adsorption column CA2. in the collection tube. Repeat this step 2 times.

5. Centrifuge the adsorption column at 12,000 rpm for 2 min to remove as much rinse solution as possible. The adsorption column CA2 was opened and placed at room temperature for 10 min.

6. Put the adsorption column CA2 into a 1.5ml low adsorption EP tube, add 50u1 of elution buffer EB dropwise to the middle of the adsorption membrane, place it at room temperature for 2min, and centrifuge at 12000rpm for 2min to collect the DNA solution. The solution obtained by centrifugation was added back to the centrifugal adsorption column, placed at room temperature for 2 minutes, centrifuged at 12,000 rpm for 2 minutes, and the DNA solution was collected into a centrifuge tube. The 4 collection tubes were shaken and centrifuged, and the solution in 3 collection tubes was transferred to another collection tube with a low adsorption pipette tip, shaken for 30 s, centrifuged briefly, and temporarily stored at 4°C.

2.2.2.5 Dilution

The purified product is precisely quantified and diluted to the concentration required for sequencing.

2.2.3 High-throughput sequencing

2.2.3.1 Bridge PCR

1. Denature the double-stranded DNA library to single-stranded using NaOH.

2. Hybridize the single-stranded DNA template to the Flow Cell.

3. Synthesize the first strand using oligos on the surface of the Flow Cell as a primer.

4. The single-stranded DNA template was washed away, and a 35-cycle bridge PCR was performed with the synthesized first strand as the template.

5. Remove the DNA strand attached to the P5 adapter from the Flow Cell.

6. Blocking the 3'-OH prevents further extension of the DNA strand during subsequent sequencing.

7. Hybridization sequencing primers.

2.2.3.2 Sequencing reaction

1. The bridge PCR products were sequenced on the Illumina X-10 sequencing platform, and the operation process was carried out according to the standard SOP.

2. Reagent preparation, sample loading, and read 1 sequencing.

3. Hybridize the Index sequencing primers to perform Index sequencing.

4.Paired End Turnround, synthesizing Read1 complementary strand.

5. Hybridize the Read 2 sequencing primer to perform Read 2 sequencing.

3. Data analysis

3.1 Multiplex PCR agarose electrophoresis

As shown in Figure 8 and Figure 9, 3% agarose gel electrophoresis was used to detect the effect of PCR products to determine whether the PCR was successful. DNALadder from bottom to top: 100, 200, 300, 400, 500bp. The electrophoresis position of the library is at 300bp+, and the quality control is qualified.

3.2 Fluorescence quantitative PCR quality control

The PCR products of the two schemes were quantified, and the homogeneity of each amplicon was examined by CT value. Figures 10 and 11 show the final optimized fluorescence quantitative PCR results of the project. The overall CT interval of the second-round products is within 5 CTs, and the quality control is qualified.

3.3 Agilent 2100 Quality Control of Libraries

After the library was purified, the quality control of the library was carried out with Agilent 2100, the main peak position was 400bp, and the quality control was qualified.

3.4 Sequencing quality control

3.4.1 Introduction to Quality Control of Sequencing Data

The NGS workflow includes nucleic acid extraction and detection, library construction and library detection, and on-machine sequencing. These links are strictly controlled to ensure the accuracy of sequencing data. After the data is downloaded, in order to ensure the accuracy of subsequent bioinformatics analysis results, it is often necessary to remove low-quality sequences or sequencing adapters through certain quality control methods.

3.4.2 Raw sequencing data

The original binary basecal ing data obtained by sequencing is converted into sequence data by Illuminabcl2fastq software, which is called PF data or Raw data in the present invention, and the result is stored in the fastq file format (file name: *.fastq.gz), and the fastq file is obtained by the user The most original file, which stores the sequence of reads and the sequencing quality of reads. Each read in the fastq format file is described by four lines:

@E00477:71:H7YJWALXX:7:1101:12236:18011:N:O:CGAGGCTG

GATCATTACTATATTTTGCTACGGTTGAGAGAAACGAATGCCCCTGTAAC

+

AAFFFAKKKKKKKKKKKKKKKKKKFFFKKKKKKKKKKKKKKKAAFKKKKK

The first line is the sequence name, generated by the sequencer, including the index sequence and the read1 or read2 flag;

Line 2 is the sequence; consists of uppercase "ACGTN";

The third line is the sequence ID, and it is also directly represented by "+" after omitting the ID name;

Row 4 is the sequencing quality of the sequence, with each letter corresponding to each base in row 2.

Among them, the conversion relationship between the ASCII code quality value and the decimal quality value is as follows:

Qscore=ASCII value-33

For example: the ASCII value corresponding to @ is 64, then the corresponding base quality value is 31 (64-33). Starting from IlluminaGAPipeline v1.8, the range of sequencing base quality values is [0,41], that is, the ASCII value is expressed as [#,K]. Concise correspondence between Illumina sequencing error rates and sequencing quality values. Specifically, if the sequencing error rate is represented by E, and the Illumina base quality value is represented by Q, the relationship is as follows: Q=-10log10(E).

3.4.3 Sequencing data yield statistics

Illumina RTA software performs image recognition and basecalling, and Illuminabcl2fastq 2.17 performs demultiplexing according to the index information of each sample and counts the number of reads and base sequencing quality (Q30).

Table 19 Sequencing sample data output statistics table (display part)

Note:

(1) Sample: sample name;

(2) Raw reads: Count the total number of double-ended Readpairs of raw data (total number of Reads);

(3) Raw data (bp): raw data yield, the number of sequenced sequences multiplied by the length of the sequenced sequence, in by;

(4) Q30 (%): Calculate the percentage of bases with Phred values greater than 30 in the total bases, and the average value of Read1 and Read2 statistics together;

(5) Clean reads: Count the total number of double-ended Readpairs of valid sequences (total number of Reads);

(6) Clean data (bp): The amount of valid data after filtering, the number of sequenced sequences after filtering is multiplied by the length of the sequenced sequence, in by.

The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention shall be included in the protection of the present invention. within the range.

Claims (10)

1. a multiplex PCR_SNP genotyping detection method, is characterized in that, during described multiplex PCR_SNP genotyping detection method PCR amplification, when the 3' end base of primer is fully matched with template, DNA polymerase carries out extension reaction, The detection signal is strong; if the base at the 3' end of the primer does not match the template, the detection signal is weak; different primers are designed according to the different genotypes of the alleles to distinguish different alleles. The 5 ends of the primers of the gene are respectively connected with a different tag sequence, and the sequence can hybridize with the tag sequence ordered on the chip to realize the interpretation of the genotype. 2. multiple PCR_SNP genotyping detection method as claimed in claim 1, is characterized in that, four SNP site design primers of described multiple PCR_SNP genotyping detection method, and connect Tag1 respectively at the 5' end of upstream primer /Tag2, Tag3/Tag4, Tag5/Tag6, Tag7/Tag8, use these four pairs of Tag to make chips at the same time, each Tag repeats 3 spots, and simultaneously spot 3 hybridization positive quality control points PC, hybridization negative quality control 3 for NC, 3 for Blank, and 3 for Hex. 3 . The multiplex PCR_SNP genotyping detection method according to claim 1 , wherein the multiplex PCR_SNP genotyping detection method comprises designing PCR amplification primers and single-base extension primers of the SNP sites to be detected. 4 . 4. multiplex PCR_SNP genotyping detection method as claimed in claim 3, is characterized in that, described multiplex PCR_SNP genotyping detection method comprises extracting DNA in blood sample, quantifying with spectrophotometer, agarose gel electrophoresis quality inspection , the concentration of DNA that has passed the quality inspection is adjusted to 50ηg/ul, and stored at -20°C for later use. 5. multiple PCR_SNP genotyping detection method as claimed in claim 4 is characterized in that, described multiple PCR_SNP genotyping detection method comprises PCR amplification, adopts multiple PCR amplification technology, and each reaction total volume 5ul, comprises Template DNA 10ng, HotstarTaq 0.5U, each amplification primer 0.5pmol, 25mM dNTP, 0.1ul, the reaction conditions are 94°C for 4 minutes; 94°C for 20 seconds, 56°C for 30 seconds, 72°C for 1 minute, 45 cycles; 72°C 3 minutes; 4°C. 6. multiple PCR_SNP genotyping detection method as claimed in claim 5, is characterized in that, described multiple PCR_SNP genotyping detection method comprises PCR product purification, and PCR reaction product uses 0.5U SAP to process, removes free dNTP in the system , the reaction system is 7 μl, including 5 μl of PCR product, 2 μl of SAP mixture, 0.5 U of SAP, 0.17 μl of buffer, and the reaction program is 37°C for 20 minutes; 85°C for 5 minutes; 4°C. 7. The multiplex PCR_SNP genotyping detection method as claimed in claim 6, wherein the multiplex PCR_SNP genotyping detection method comprises single base extension, and the total volume 9 μl reaction system comprises 7 μl of the PCR product after SAP treatment, wherein Each extension reaction primer mix 0.804l, iPLEX enzyme 0.041l, extension mix 0.2μl, the reaction program is 94°C for 30 seconds; 94°C for 5 seconds; 52°C for 5 seconds, 80°C for 5 seconds for 5 cycles; return to 94°C for 5 seconds, A total of 40 cycles; 3 min at 72°C, 4°C. 8. multiple PCR_SNP genotyping detection method as claimed in claim 7 is characterized in that, described multiple PCR_SNP genotyping detection method comprises resin purification, and each extension reaction product is purified with 6mg Clean Resin resin; Chip spotting and mass spectrometry detection, the purified product was transferred to a 384-well SpectroCHIP chip, and measured on the computer. The SpectroCHIP chip was analyzed by MALDI-TOF matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and the detection results were typed and output by software. 9. multiple PCR_SNP genotyping detection method as claimed in claim 8, is characterized in that, described multiple PCR_SNP genotyping detection method comprises: (1) Data preprocessing, filtering out non-informative sites in all samples and sites with typing success rate less than 90%; filtering out sites with MAF less than 0.01 in control samples; using control samples, for each Hardy-Weinberg equilibrium test for each SNP site; (2) Single-point association analysis method, for each locus after preprocessing, the chi-square test and Fisher's exact test were used to test the differences between the cases and controls for the four cases, and for the cases where the odds ratio could be calculated, calculate Odds ratios and their 95% confidence intervals. 10. multiple PCR_SNP genotyping detection method as claimed in claim 9, is characterized in that, described multiple PCR_SNP genotyping detection method comprises four kinds of situations: (1) Perform chi-square test and Fisher exact test on allele frequencies and calculate OR value and 95% confidence interval; (2) Chi-square test and Fisher's exact test for genotype frequencies; (3) Chi-square test and Fisher's exact test were performed on the genotype frequencies for the dominant model, and the OR value and 95% confidence interval were calculated; (4) Chi-square test and Fisher's exact test were performed on the genotype frequencies for the recessive model, and the OR value and 95% confidence interval were calculated.

Images