Method for building genome sequencing library, corresponding joint sequence and kit

A genome sequencing and linker sequence technology, which is applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve the problems of low double link link connection efficiency, high connection efficiency, increased steps and reagent costs, etc., which is not easy to achieve. The effect of connector self-connection, efficient connection and cost advantage

Inactive Publication Date: 2018-11-06
GUANGZHOU MICROCHIP BIOTECH CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main advantages and disadvantages of these three kinds of joints are that the efficiency of double-linked joints with flat ends is moderate, and it is easy to form joint self-connection; the connection efficiency of double-linked joints with Y-shaped structure is low, and an additional annealing step is required for the manufacturing process, and two steps before annealing The concentration and ratio of single strands are often unbalanced and unstable due to quantitative problems; the U-shaped structure of the single-chain linker is a single-chain structure, which naturally forms a partially double-stranded stem-loop structure without additional annealing. High efficiency, not easy to generate adapter self-ligation, but need to use enzymes to open the U-shaped structure before PCR amplification of the library, increasing the experimental steps and reagent costs

Method used

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  • Method for building genome sequencing library, corresponding joint sequence and kit
  • Method for building genome sequencing library, corresponding joint sequence and kit
  • Method for building genome sequencing library, corresponding joint sequence and kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The length of embodiment 1 carbon chain modification

[0037] 1. Cell culture

[0038]293T cells were cultured at 37°C and 5% CO2 using GIBCO's DMEM and 10% FBS serum. After 2 days of normal culture, the cell density was around 60-80% (40,000-50,000).

[0039] 2. Genomic DNA extraction

[0040] Genomic DNA was extracted according to Qiagen's QIAamp DNAMini Kit instructions. 3. Genome sequencing library construction 100ng initial amount (cell DNA), using NEBNext DNA Ultra-fast Library Preparation Kit-Illumina for library construction, the main steps are: DNA fragmentation (the average fragment length after fragmentation is 250bp); Repair and purification; A at the 3' end and purification; sequencing linker ligation and purification; PCR amplification and purification, and a genome sequencing library was obtained. The specific steps were carried out according to the instructions of the ultra-rapid library preparation kit. The sequences of sequencing adapters modified w...

Embodiment 2

[0049] Example 2 The number of units modified by the carbon chain

[0050] 1. Cell culture

[0051] Concrete method is with embodiment 1.

[0052] 2. Genomic DNA extraction

[0053] Concrete method is with embodiment 1.

[0054] 3. Genome sequencing library construction

[0055] Concrete method is with embodiment 1.

[0056] The specific steps were performed according to the instructions of the ultra-rapid library preparation kit. The sequencing linker sequences with different numbers of long carbon chain modification units are shown in Table 4, and the concentration of the storage solution for the ligation reaction was 15 μM.

[0057] Table 4

[0058]

[0059] 4. Genome sequencing library quality inspection

[0060] Concrete method is with embodiment 1.

[0061] 5. Result analysis:

[0062] The quality inspection results of the genome sequencing library showed that, using NEB adapters as positive controls, 1 C18 (18 continuous long carbon chain modifications), 2 C1...

Embodiment 3

[0066] Embodiment 3 linker concentration

[0067] 1. Cell culture

[0068] Concrete method is with embodiment 1.

[0069] 2. Genomic DNA extraction

[0070] Concrete method is with embodiment 1.

[0071] 3. Genome sequencing library construction

[0072] Concrete method is with embodiment 1.

[0073] The specific steps were performed according to the instructions of the ultra-rapid library preparation kit. The sequences of the sequencing adapters are shown in Table 6, and the concentrations of the stock solution for the ligation reaction were 5 μM, 10 μM, 15 μM and 20 μM, respectively.

[0074] Table 6

[0075]

[0076] 4. Genome sequencing library quality inspection

[0077] Concrete method is with embodiment 1.

[0078] 5. Result analysis

[0079] The quality inspection results of the genome sequencing library showed that, using the NEB adapter as a positive control, using the C18 (18 continuous long carbon chain modification) sequencing adapter, and different seq...

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Abstract

The invention discloses a method for building a genome sequencing library, a corresponding joint sequence and a kit; The joint sequence used for building the genome sequencing library ingeniously usesthe chemical modification of C3-C21 long carbon chains; the chemical modification is applied to a stem loop structure sequencing joint formed by single chains. Compared with the existing mainstream library building joint, the joint has the advantages that the joint dimerization cannot be easily generated; the additional double-chain annealing is not needed; the use of enzymes for opening loop structures in the stem loops is not needed; on the basis of not influencing the later stage PCR amplification, the reaction time is greatly saved, and the reagent cost is greatly reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for constructing a genome sequencing library, a corresponding linker sequence and a kit. Background technique [0002] With the development of modern science and technology, the research of life science has entered the era of omics. Gene and genome sequencing technology has become an indispensable means in modern life science research, especially in genomics research. In recent years, the rapid development of next-generation genome sequencing technology has brought about unprecedented prosperity in genomics research. Next-generation sequencing technology has been widely used in various fields of life sciences, agronomy, medicine, environmental protection, forensic science and other fields. In next-generation sequencing, genome sequencing is the most widely used sequencing method, including genome de novo sequencing of species, re-sequencing of individuals or va...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869C40B50/06C12N15/10C12N15/11
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2525/191
Inventor 曹亮束文圣吴胜标王璋郝祎琪
Owner GUANGZHOU MICROCHIP BIOTECH CO LTD
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