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A method and kit for rapidly constructing a single-cell DNA sequencing library

A DNA sequencing and single-cell technology, applied in the field of construction of single-cell DNA sequencing library, can solve problems such as complex process, large base bias of PCR, poor genome coverage and uniformity

Active Publication Date: 2021-04-06
HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The above-mentioned single-cell DNA sequencing library construction methods and kits mainly have two defects: First, the process from WGA to library construction is complicated and takes a long time. It takes at least two days to complete the entire process, which is not suitable for Large-scale production applications; secondly, the PCR base bias introduced by WGA amplification is relatively large, resulting in poor coverage and uniformity of the entire genome

Method used

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  • A method and kit for rapidly constructing a single-cell DNA sequencing library
  • A method and kit for rapidly constructing a single-cell DNA sequencing library
  • A method and kit for rapidly constructing a single-cell DNA sequencing library

Examples

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Comparison scheme
Effect test

Embodiment 1

[0078] The construction method of the single-cell DNA sequencing library of Example 1 mainly includes the following steps (see image 3 ):

[0079] (1) Cell lysis: This step can be performed using any protease, protein denaturing reagent and lysis buffer system known to those skilled in the art that are suitable for cell lysis.

[0080] (2) Pre-amplification of genomic DNA can be performed using any thermostable DNA polymerase known to those skilled in the art, for example: LA-Taq, rTaq, Phusion, Deep Vent, Deep Vent (exo-), Gold 360 , PlatinumTaq, KAPA 2G Robust.

[0081] The preamplification primers used in the present invention are mainly composed of three parts: a common region, which has different sequences in different sequencing platforms; a degenerate base region, which only contains two kinds of bases, A and C, or A and G , or T and C, or T and G; a region of random degenerate bases, where each base can be A, T, C, or G, and two sulfurs are added between the last th...

Embodiment 2

[0114] The library construction method in Example 2 is basically the same as in Example 1, the difference is that in Example 2, the starting sample is multi-cell rather than single-cell (please refer to image 3 ).

[0115] A specific example of performing multicellular DNA sample sequencing according to the method of Embodiment 2 of the present invention is shown below

[0116] Step 1: Multiple cell lysis. Prepare cell lysis buffer as shown in Table 1, incubate at 60°C for 20 minutes, 95°C for 4 minutes, then keep samples at 4°C.

[0117] Table 1

[0118]

[0119] Step 2: Genomic DNA pre-amplification. Prepare the pre-amplification PCR system shown in Table 2.

[0120] Table 2

[0121]

[0122] The PCR reaction scheme is: a.95°C for 3 minutes→b.16 cycles as follows: 98°C for 20 seconds, 15°C for 50 seconds, 25°C for 40 seconds, 35°C for 30 seconds, 65°C for 40 seconds, 72°C for 1 minute→ c. Keep at 4°C.

[0123] Step 3: Secondary amplification of genomic DNA. Pr...

Embodiment 3

[0132] The library construction method in Example 3 is basically the same as in Example 1, except that in Example 3, the starting sample is DNA instead of cells. In this example, the starting sample can be genomic DNA, mitochondrial DNA, long PCR products, or long chromatin-precipitated DNA (see image 3 ).

[0133] A specific example of performing DNA sample sequencing according to the method of Embodiment 3 of the present invention is shown below

[0134] Step 1: Fragmentation of long fragments of DNA. Prepare cell lysis buffer as shown in Table 1, incubate at 60°C for 20 minutes, 95°C for 4 minutes, then keep samples at 4°C. Alternatively, DNA fragments were directly incubated at 95°C for 4 minutes, and then the samples were kept at 4°C.

[0135] Table 1

[0136]

[0137] Step 2: DNA pre-amplification. Prepare the pre-amplification PCR system shown in Table 2.

[0138] Table 2

[0139]

[0140] The PCR reaction scheme is: a.95°C for 3 minutes→b.16 cycles as follo...

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Abstract

The present invention relates to a method and a kit for constructing a single-cell DNA sequencing library, and more specifically, the present invention relates to a method and a kit for constructing a single-cell DNA sequencing library for high-throughput sequencing.

Description

technical field [0001] The present invention relates to a method and a kit for constructing a single-cell DNA sequencing library, and more specifically, the present invention relates to a method and a kit for constructing a single-cell DNA sequencing library for high-throughput sequencing. Background technique [0002] With the completion of the Human Genome Project, Sanger sequencing has entered the research of many researchers. However, the continuous innovation of technology has made this dideoxynucleotide-terminated sequencing mode increasingly unable to meet the needs of sequencing, so a new high-throughput sequencing (also called second-generation sequencing) mode emerged as the times require. This sequencing technology enables lower sequencing costs, higher sequencing throughput, and shorter sequencing time. The core idea of ​​high-throughput sequencing technology is sequencing-by-synthesis, that is, to determine the sequence of DNA by capturing the markers of newly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12N15/11
Inventor 石燕滨吴凯王文璐张建光
Owner HANGZHOU BERRYGENOMICS GENE DIAGNOSIS TECH
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