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Simultaneous single-cell genome and transcriptome library construction and sequencing methods Sequencing methods and applications based on single-cell integrative genomics

A genomics, single-cell technology, applied in the field of high-throughput sequencing, can solve the problems of inability to display the whole picture of a single cell, imperfection, etc.

Inactive Publication Date: 2018-12-07
SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, all the research results related to single-cell technology are still incomplete, and they are divided into two categories: a) only measure the genome, not the transcriptome; b only measure the transcriptome, not the genome; however, these analysis methods cannot show the whole picture of single cells

Method used

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  • Simultaneous single-cell genome and transcriptome library construction and sequencing methods Sequencing methods and applications based on single-cell integrative genomics
  • Simultaneous single-cell genome and transcriptome library construction and sequencing methods Sequencing methods and applications based on single-cell integrative genomics
  • Simultaneous single-cell genome and transcriptome library construction and sequencing methods Sequencing methods and applications based on single-cell integrative genomics

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1: nucleoplasm separation

[0064] 1) Take a single mouse secondary oocyte;

[0065] 2) Separation of nucleus and cytoplasm:

[0066] Using an eppendorf micromanipulator, the nuclei and cytoplasm of single cells were separated by microinjection (Microinjection) method. Prepare a glass tube with an aperture size close to that of a single secondary oocyte and a single nucleus (in the microinjection method, the diameter of the microcapillary needle is 0.5-5 microns, and the holding needle ) with a diameter of 10-50 microns (microns)), after the separation, the nucleus and cytoplasm were placed in different PCR tubes, then stored in liquid nitrogen, and the next one was treated in the same way. (Note: For the instability of RNA in the cytoplasm, we will inject a small amount of RNA inhibitors into the cytoplasm during the membrane penetration step to prevent RNA degradation during membrane rupture and nuclear division. At the same time, ensure that the entire proc...

Embodiment 2

[0067] Example 2: Construction of whole genome and whole transcriptome high-throughput sequencing libraries, and high-throughput sequencing

[0068] 1) Whole-genome amplification of a single nucleus

[0069] Since the amount of DNA in a single cell is very limited (about 6pg), the amount of DNA in a single cell should be uniformly amplified before sequencing. We used the GenomePlex WGA4 amplification method (Sigma-Aldrich Single Cell Whole Genome Amplification Kit (Cat. No. WGA4-50RXN, refer to the kit manual for specific steps), this method randomly fragments the genome through a short-term high-temperature operation to form a series of short templates, and then randomly fragments these short-strand DNA Annealing, adding a library composed of specific sequences on both sides of each short chain, and then performing isothermal initial amplification for these specific sequences.

[0070] 2) Single cell enucleated cytoplasm whole transcriptome amplification and library constr...

Embodiment 3

[0076] Embodiment 3: bioinformatics analysis

[0077] Sequences from the genome sequence and mRNA fastq files were aligned with the genome using the Bowtie and Tophat methods, respectively [72,73]. And use the Varscan method to find out the difference between the genome and mRNA [38]. The default setting of varscan is to cover at least 8 sequence numbers before being used for subsequent analysis, and the minimum variant allele frequency is 0.01. A variant with an allele frequency of less than 75% is called a heterozygote, otherwise a homozygous variant is assigned. RESs were detected by aligning genomic sequences with mRNAfastq file sequences, covering at least 8 sequences per locus. The FPKM value in the Cufflinks method was used to determine the expression level of the gene [74]. In the downloaded Ensembl gene annotation (10mm), only protein-coding genes and intergenic long non-coding RNA (large intergenic non-coding RNA, lincRNA) were selected [75].

[0078] Sequencing ...

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Abstract

The invention provides a method for high-throughput library construction and sequencing for both a single cell genome and transcriptome, and further provides a sequencing method based on the single cell integrated genomics (SCIG), and application of the sequencing method. Through the SCIG scheme provided by the invention, both the single cell genome and transcriptome can be obtained in a single experiment, and genetic and epigenetic information in a single cell can be obtained for comprehensive analysis, so as to achieve all-dimensional and multi-level state display of the single cell; therefore, the SCIG has superiority in detection of the general and unique characteristics of the single cell.

Description

technical field [0001] The invention relates to the field of high-throughput sequencing, in particular to a method for simultaneously constructing and sequencing single-cell genomes and transcriptome libraries, and a sequencing method and application based on single-cell integrated genomics (SCIG). Background technique [0002] Single-cell DNA whole-genome sequencing and RNA transcriptome sequencing technologies have developed rapidly in recent years. The single-cell DNA amplification method, based on the original Multiple displacement amplification (MDA) method, has successively produced the GenomePlex (WGA4) method and the Multiple Annealing and Looping Based AmplificationCycles (MALBAC) method. In terms of single-cell mRNA amplification, new technologies such as SMART-Seq, CEL-Seq, and Quartz-Seq have also been successively produced, which greatly improve the amplification efficiency of trace amounts of mRNA. [0003] At present, all the research results related to singl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06C12Q1/6869
Inventor 李周芳贺建奎王嫣郭佳杰张萌
Owner SOUTH UNIVERSITY OF SCIENCE AND TECHNOLOGY OF CHINA
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