Single cell whole genome sequencing method

A whole-genome sequencing and single-cell technology, applied in the field of molecular biology, can solve problems such as the application of single-molecule sequencing and single-cell whole-genome sequencing.

Inactive Publication Date: 2020-07-03
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Application of single-molecule sequencing to s...

Method used

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Examples

Experimental program
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Effect test

Embodiment 1

[0094] Embodiment 1 Amplification method of the present invention Amplifies single cell genome and sequencing analysis

[0095] See the experimental procedure figure 1 , the specific experimental steps and results are as follows:

[0096] 1. Obtain single cells

[0097] (1) K562 (human chronic myeloid leukemia cell line) cells were revived and cultured to 10 6 / mL.

[0098] (2) Use a capillary to pick a single cell into a PCR tube.

[0099] 2. Extract single-cell genome and amplify it

[0100] An amplification system was prepared using a single cell expansion kit (REPLI-g single cell kit (Qiagen, Cat. No. 150343)).

[0101] (1) Take 33 μL Reconstituted Buffer DLB and add 3 μL dithiothreitol (DTT(1M)) to configure buffer D2.

[0102] (2) Add 3 μL buffer D2 to 4 μL single cell suspension, mix gently, and centrifuge.

[0103] (3) Incubate at 65°C for 10 minutes.

[0104] (4) Add 3 μL of stop solution, mix gently, and centrifuge to obtain a single cell lysate.

[0105] (5...

Embodiment 2

[0119] Example 2 The microdroplet-based amplification method of the present invention amplifies single-cell genome and sequence analysis

[0120] See the experimental procedure Figure 6 , the specific experimental steps and results are as follows:

[0121] 1. Obtain single cells

[0122] (1) K562 (human chronic myeloid leukemia cell line) cells were revived and cultured to 10 6 / mL.

[0123] (2) Use a capillary to pick a single cell into a PCR tube.

[0124] 2. Extract single-cell genome and amplify it

[0125] An amplification system was prepared using a single-cell genome amplification kit (REPLI-gsinglecellkit, Qiagen, Cat. No. 150343).

[0126] (1) Take 33 μL Reconstituted Buffer DLB and add 3 μL dithiothreitol (DTT, 1M) to configure buffer D2.

[0127] (2) Add 3 μL buffer D2 to 4 μL single cell suspension, mix gently, and centrifuge.

[0128] (3) Incubate at 65°C for 10 minutes.

[0129] (4) Add 3 μL of stop solution, mix gently, and centrifuge to obtain a single...

Embodiment 3

[0148] Embodiment 3 comparative experiment

[0149] 1. Bulk genomic DNA extraction.

[0150] 1.1 K562 (human chronic myeloid leukemia cell line) cells were revived and cultured to 10 6 / mL.

[0151] 1.2 Take 2×10 6 cells, and the genomic DNA was extracted by the spin column method. The total DNA mass was 20 μg detected by absorbance (nanodrop). 1% agarose gel electrophoresis running gel results see Figure 10 .

[0152] 2. Send the bulk genomic DNA to Wuhan Future Group Company for sequencing.

[0153] 3. The results of bulk genome DNA sequencing are considered as the original sequence before amplification and serve as the genome reference sequence. Comparing the original sequence with the sequencing results of the single-cell amplification method and the microdroplet-based amplification method can evaluate the probability of base mutation and chromosome structure variation during the amplification process of the two single-cell genome amplification methods , so as to ju...

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Abstract

The present invention provides a single cell whole genome sequencing method and an application of an amplification method in constructing a gene library and conducting single molecule sequencing. Theamplification method comprises step of contacting nucleic acid with nucleic acid polymerase and random primers to carry out multiple displacement amplification of the nucleic acid and strands.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a single-cell whole-genome sequencing method and the application of an amplification method in single-molecule sequencing. Background technique [0002] Single-cell whole-genome sequencing technology is a new technology for amplifying and sequencing the whole genome at the single-cell level. The principle is to amplify the micro-volume whole-genome DNA of isolated single cells, obtain a complete genome with high coverage, and then use exon capture and high-throughput sequencing to reveal cell population differences and cell evolution relationships. [0003] Single-cell whole-genome amplification refers to a new technology for amplifying the whole genome at the single-cell level. Throughput sequencing for revealing cellular heterogeneity. Currently, WGA methods mainly include primer extension pre-amplification PCR (PEP-PCR), degenerate oligonucleotide primer PCR (degenerate olig...

Claims

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Application Information

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IPC IPC(8): C12Q1/6869C12Q1/6806
CPCC12Q1/6869C12Q1/6806
Inventor 白净卫刘册
Owner TSINGHUA UNIV
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