A kind of preparation method of next generation sequencing library
A sequencing library, a next-generation technology, applied in the field of preparation of next-generation sequencing libraries, can solve problems such as cost reduction, and achieve the effect of reducing workload
Active Publication Date: 2022-07-26
深圳中农京跃生物技术有限公司
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Problems solved by technology
[0002] In October 2015, as Illumina announced the HiSeq X TM After the application of the sequencing system was expanded to the whole genome sequencing of non-human species, the cost of next-generation sequencing (Next-Generation Sequencing, NGS) dropped significantly; The new sequencing system NovaSeq was released at the conference TM series instrument, and claims that the instrument costs less reagent per run than the HiSeq X TM 30% lower, but data output is HiSeq X TM 3 times that of human genome sequencing, further reducing the price of human genome sequencing to $100 in the future
In this context, conventional sequencing library construction methods have become the bottleneck step in the next step of large-scale population resequencing projects
Method used
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Embodiment 1
[0028] Taking the library construction process of 30 rice genomic DNAs as an example, the agarose gel electrophoresis detection results of the rice DNAs used are as follows: figure 1 As shown (part of the sample), all DNA bands are complete and clear, and the quality meets the library construction requirements. The operation steps are as follows:
[0029] 1. Quantification of DNA
[0030] 1.1 Use agarose gel electrophoresis to perform quality detection on rice genomic DNA, and the results of DNA quality detection in the present embodiment are as follows: figure 1 .
[0031] 1.2 Qubit quantitative steps
[0032] The dsDNA High Sensitivity Kit measures the concentration of DNA. In the present embodiment, the concentration of 30 parts of rice DNA is shown in Table 1:
[0033] Table 1
[0034]
[0035] 2. DNA adapter sequences required for ligation and sequencing at both ends of the DNA fragment to be tested
[0036] Add the DNA adapter sequences required for sequencin...
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The invention discloses a preparation method of a next-generation sequencing library. Carry out the following steps: DNA quantification, adding DNA adapter sequences required for sequencing at both ends of the DNA fragment to be tested, detecting the concentration of the DNA library and the distribution pattern of DNA fragment length, calculating and mixing the mixing ratio of the DNA library, and mixing the library Fragment selection, detection and quantification of selected DNA libraries, and sequencing of selected DNA libraries. In the method of the present invention, multiple libraries are mixed together after PCR to perform fragment sorting and Q-PCR quantification, which greatly reduces the workload, and the distribution of the final sequencing data volume in different samples is not significantly affected.
Description
technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing a next-generation sequencing library. Background technique [0002] In October 2015, with the announcement of Illumina's HiSeq X TM After the application of sequencing systems is extended to whole genome sequencing of non-human species, the cost of Next-Generation Sequencing (NGS) has dropped significantly; on January 9, 2017, the sequencing giant Illumina Company was in the famous JP Morgan Health. NovaSeq, a new sequencing system, was released at the conference TM series instrument and claim that the instrument costs more reagents per run than the HiSeq X TM 30% lower, but data throughput is HiSeq X TM 3 times the price of human genome sequencing in the future, further reducing the price of human genome sequencing to $100. HiSeq X TM and NovaSeq TM The launch of , also enables large-scale population resequencing projects for numerous species...
Claims
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IPC IPC(8): C12Q1/6806C12N15/10C40B50/06
CPCC12N15/1093C12Q1/6806C40B50/06C12Q2525/191C12Q2531/113
Inventor 常玉晓穆建强赵胜卢颖刘可
Owner 深圳中农京跃生物技术有限公司