Simplified library construction method and reagent

A library construction and fragmentation technology, applied in the field of simplified library construction methods and reagents, can solve the problems of inability to integrate the fragmentation and end repair steps, increase the loss of DNA samples, and take a long time to meet the requirements of sequencing on-machine, The effect of reducing DNA loss

Active Publication Date: 2018-07-27
CAPITALBIO GENOMICS
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It can be seen that the existing library construction steps are cumbersome, it takes a long time to purify multiple magnetic beads, fragmentation and end repair steps cannot be integrated, and adapter ligation products need to be further enriched. These processes are not only costly and time-consuming, but also multi-step Purification inevitably increases DNA sample loss

Method used

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  • Simplified library construction method and reagent
  • Simplified library construction method and reagent
  • Simplified library construction method and reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1, a kind of simplified library construction method

[0040] A simplified library construction method of the present invention, the specific process is as follows:

[0041] (1) Fragmentation and end repair

[0042] Prepare reaction solution A (10X) for fragmentation and end-repair one-step reaction, the formula is as follows:

[0043]

[0044]

[0045] Configure the following system:

[0046]

[0047] Wherein, enzyme mixture B includes fragmentation enzyme (NEB Company) and end repair enzyme (Life Company), and the two are mixed at a volume ratio of 1:1.

[0048] After mixing, carry out the following reaction, and store at 4°C after the reaction:

[0049]

[0050] (2) Ligation of sequencing adapters

[0051] For the adapter ligation of the product after the previous step reaction, add the following reagents:

[0052]

[0053] Among them, the P1 adapter is a P1-Adapter composed of the following two positive and negative sequences:

[0054]...

Embodiment 2

[0061] A simplified library construction method, after the step (1) in Example 1, the "magnetic bead purification" step is added, and other steps remain unchanged. Among them, the supplementary magnetic bead purification steps are as follows: Purify the fragmented and end-repaired product with 36 μL of XP magnetic beads for 5 minutes, place it on the magnetic stand for 3 minutes, then aspirate the liquid, wash twice with 500 μL of 75% ethanol, and blot the residue. Dry the liquid, add 20 μL Low TE solution, mix well, let it stand for 5 minutes, absorb the liquid, and it will be the purified fragmentation and end repair product, which is used to connect the sequencing adapter.

Embodiment 3

[0063] A simplified library construction method. In step (1) of Example 1, the formula of reaction solution A (10X) is optimized. The formulas of each group are as follows, and other steps remain unchanged:

[0064]

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Abstract

The invention discloses a simplified library construction method and a reagent. The construction method and the reagent, disclosed by the invention, can finish fragmentation and final segment repairing in one step; a product obtained by the fragmentation and the final segment repairing can be not purified and a library enrichment step is omitted in a whole library construction method; a sequencinglibrary, which is finally obtained, can meet sequencing online requirements, and DNA (Deoxyribonucleic Acid) loss caused by multi-step purification is also reduced; a detection result of a sample isnot influenced.

Description

technical field [0001] The invention belongs to the field of gene sequencing, and more specifically relates to a simplified library construction method and reagents. Background technique [0002] With the development of sequencing technology, genome sequencing has become a common technical means. In the past, genome sequencing was mostly performed at the multi-cell level. In recent years, single-cell genome sequencing of circulating tumor cells and preimplantation screening cells has become a hot spot. For these single-cell genome sequencing, the existing The sequencing method is mainly to amplify the whole genome of a single cell to increase the amount of templates to meet the needs of a high-throughput sequencing platform, then build a library of the amplified whole-genome DNA, and finally perform high-throughput sequencing and analysis on the library. [0003] For the library construction step, the process of the prior art mainly includes the following steps: ① Fragmenta...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12Q1/6869
CPCC12N15/1093C12Q1/6869C40B50/06C12Q2527/125C12Q2535/122C12Q2525/191
Inventor 糜庆丰饶兴蔷陈嘉欣罗东红彭春方陈样宜黄铨飞
Owner CAPITALBIO GENOMICS
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