Method and kit for constructing simplified genome library
A genome library and kit technology, applied in the field of high-throughput sequencing library construction, can solve the problems of cumbersome steps and high cost, and achieve the effects of simple and easy steps, low cost and complicated solution steps.
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Embodiment 1
[0061] 1. Extraction of genomic DNA to be tested
[0062] Genomic DNA of Nipponbare individual rice leaves was extracted as the object of library construction in this example. The extraction operation is briefly described as follows:
[0063] 1.1 Take 100mg of fresh leaf tissue, add liquid nitrogen and grind thoroughly;
[0064] 1.2 Use Tiangen Plant Genomic DNA Extraction Kit or CTAB method to extract;
[0065] 1.3 Extracted DNA for quality inspection.
[0066] 2. PCR amplification enrichment target region (PCR-1)
[0067] 1. Primer Design
[0068] 1) At the 5'->3 Design unique bridging primers, tag sequences and non-specific sequences at the 'end;
[0069] 2) See Table 2 for specific primer sequence information.
[0070] Table 2:
[0071]
[0072] 2. PCR amplification
[0073] 1) Add different primer pairs to the sample genomic DNA for PCR amplification, and add a negative control CK;
[0074] 2) KODFX 20 μL reaction system was used for PCR;
[0075]
[0076...
Embodiment 2
[0119] The only difference between the operation steps of Example 2 and Example 1 lies in the procedure of the first round of PCR reaction. details as follows:
[0120] a.1×(94℃, 2min)
[0121] b.2×(98℃, 10s; 15℃ , 2min; jump to 68°C (at a heating rate of 0.8°C / s))
[0122] c.1×(68℃, 2min)
[0123] d.35×(98℃, 10s; 58℃, 30s; 68℃, 1min)
[0124] e.1×(68℃, 5min)
[0125] The DNA library obtained by the above two-step library construction method was sequenced through the Hiseq sequencing platform of Illumina Company, and the data volume of each library was measured 1Gb. The results of data analysis are as follows:
[0126] Q20, Q30: It can be seen from the figure that Pool Rice's Q20 and Q30 reached more than 94%, so the sequencing quality of this time is good.
Embodiment 3
[0128] One, DNA extraction step (same as embodiment 1)
[0129] 2. PCR amplification enrichment target region (PCR-1):
[0130] 1. Use the primers shown in Table 3, the same reaction system and PCR reaction program as in Example 1 to carry out the first round of PCR reaction.
[0131] table 3:
[0132]
[0133] 1) In forward primers (2-1F, 2-2F, 2-3F, 2-4F, 2-5F) and reverse primers (2-1R, 2-2R, 2-3R, 2-4R, 2- Design unique bridging primers, tag sequences and non-specific sequences at the 5' to 3' end of 5R);
[0134] 3. The second round of PCR
[0135] The 6 PCR products of the first round were mixed in equal amounts to form a mixed sample, and the mixed sample was subjected to the second round of PCR (PCR reaction conditions were the same as in Example 1).
[0136] 4. Library library check and computer (consistent with embodiment 1).
[0137] The results of the analysis of the sequencing data are as follows:
[0138] Q20, Q30: It can be seen from the figure that Poo...
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