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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: 2018-11-23
AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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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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  • Preparation method of next-generation sequencing library

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Embodiment 1

[0028] Taking the library construction process of 30 copies of rice genomic DNA as an example, the results of agarose gel electrophoresis of the rice DNA used are as follows: figure 1 As shown (some samples), 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 Utilize agarose gel electrophoresis to carry out quality detection to rice genome DNA, the result of DNA quality detection in the present embodiment is 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. The DNA linker sequence required for sequencing at both ends of the DNA fragment to be tested

[0036] Use transposase to add the DNA linker sequence required for sequencing...

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Abstract

The invention discloses a preparation method of a next-generation sequencing library. The preparation method is carried out according to the following steps: quantifying DNA (Deoxyribonucleic Acid); adding a needed DNA linker sequence in a sequencing process at two ends of a DNA fragment to be detected; detecting the concentration of a DNA library and a DNA fragment length distribution mode; calculating a mixing ratio of the DNA library and mixing; selecting a fragment of a mixed library; detecting and quantifying the selected DNA library; sequencing the selected DNA library. According to themethod disclosed by the invention, a plurality of libraries are mixed together after being subjected to PCR (Polymerase Chain Reaction) and are subjected to primary fragment sorting and Q-PCR (Quantitative-Polymerase Chain Reaction) quantifying, so that the working amount is greatly reduced; the distribution of a final sequencing data quantity in different samples is not remarkably influenced.

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, 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, which will further reduce the price of human genome sequencing to $100 in the future. HiSeq X TM and NovaSeq TM The launch of , also makes large-scale population resequencing projects possible for many species. However, compared with the rapid progress of sequencing technolog...

Claims

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Application Information

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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 AGRI GENOME INST OF SHENZHEN CHINESE ACADEMY OF AGRI SCI
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