High-throughput single-cell sequencing library construction method

A single-cell sequencing and library construction technology, applied in the field of single-cell sequencing library construction, can solve the problems of incompletely established technology for single-cell whole transcriptome analysis of prokaryotic microorganisms, low total concentration of RNA molecules, and short RNA half-life, etc. Improve the efficiency of joint connection, high accuracy, and reduce the false positive rate

Inactive Publication Date: 2018-12-07
天津迈基生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The difficulty in analyzing microbial cells is that most microorganisms, such as bacteria and archaeal cells, have a cell outer membrane structure that is difficult to effectively lyse; prokaryotic microorganisms have short RNA half-lives and low stability; in addition, the cell size is much smaller than that of mammals cells (10-30 picograms), so the total concentration of RNA molecules in cells is very low, about 4-10 ficks per cell, this RNA concentration is much lower than the RNA concentration in eukaryotic cells, which is RNA Extraction and analysis present many challenges
Although the whole-transcriptome analysis technology for single-cell eukaryotes has been established, the technology for whole-transcriptome analysis of single-cell prokaryotic microorganisms has not been fully established.

Method used

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Examples

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Effect test

Embodiment 1

[0022] A high-throughput single-cell sequencing library construction method, comprising the following steps,

[0023] S1, contacting the nucleic acid from the single cell with a first substance and a polymerase comprising a strand displacement activity, the contact occurs at a temperature of 4° C., a primer anneals to the nucleic acid, and the primer is extended by enzyme cleavage, Wherein said first substance comprises G rich in 30% or C rich in 30%; and comprises a restriction endonuclease site, thereby producing a mixture comprising a nucleic acid template for primer annealing; Two or more rounds of extension, melting, and annealing steps, thereby producing a mixture of nucleic acid templates, linearly generated half-amplicons, and non-linearly generated full amplicons; Under the condition that the two ends of the two ends can anneal to each other, thereby producing a circularized full amplicon; the circularized full amplicon is exposed to a restriction endonuclease, so tha...

Embodiment 2

[0027] A high-throughput single-cell sequencing library construction method, comprising the following steps,

[0028] S1, contacting the nucleic acid from the single cell with a first substance and a polymerase comprising a strand displacement activity, the contact occurs at a temperature of 20° C., a primer anneals to the nucleic acid, and the primer is extended by enzyme cleavage, Wherein said first substance comprises G rich in 35% or C rich in 35%; and comprises a restriction endonuclease site, thereby producing a mixture comprising a nucleic acid template for primer annealing; Two or more rounds of extension, melting, and annealing steps, thereby producing a mixture of nucleic acid templates, linearly generated half-amplicons, and non-linearly generated full amplicons; Under the condition that the two ends of the two ends can anneal to each other, thereby producing a circularized full amplicon; the circularized full amplicon is exposed to a restriction endonuclease, so th...

Embodiment 3

[0032] A high-throughput single-cell sequencing library construction method, comprising the following steps,

[0033] S1, contacting the nucleic acid from the single cell with a first substance and a polymerase comprising a strand displacement activity, the contact occurs at a temperature of 25° C., a primer anneals to the nucleic acid, and the primer is extended by enzyme cleavage, Wherein said first substance comprises G rich in 40% or C rich in 40%; and comprises a restriction endonuclease site, thereby producing a mixture comprising a nucleic acid template for primer annealing; Two or more rounds of extension, melting, and annealing steps, thereby producing a mixture of nucleic acid templates, linearly generated half-amplicons, and non-linearly generated full amplicons; Under the condition that the two ends of the two ends can anneal to each other, thereby producing a circularized full amplicon; the circularized full amplicon is exposed to a restriction endonuclease, so th...

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Abstract

The invention discloses a high-throughput single-cell sequencing library construction method, which comprises the following steps: S1. leading nucleic acid from a single cell to be contacted with a first substance and chain substitute activity-containing polymerase, wherein contact occurs under the condition of the temperature ranging from 4 DEG C to about 25 DEG C; annealing a primer and the nucleic acid, wherein the primer is extended through digestion; and placing the mixture under the condition that the two ends of a total amplicon can be annealed, so that the total amplicon cannot be annealed together with the first substance or a second substance, wherein the second substance contains 30%-40% of G or C; the first substance or the second substance and a semi-amplicon generated by remaining linearity in the mixture can be annealed and extended, wherein the annealing and extension can occur under the condition that nucleic acid cannot be further unwound, and therefore, a total amplicon A generated due to linearity can be generated. The construction method is smart in design and reasonable, and good in effect, high in accuracy for high-throughput sequencing library, and suitablefor population.

Description

technical field [0001] The invention relates to the technical field of single-cell sequencing library construction, in particular to a high-throughput single-cell sequencing library construction method. Background technique [0002] Non-culture-based single-cell genomics techniques have been successfully implemented and applied to various microbial ecology studies. The difficulty in analyzing microbial cells is that most microorganisms, such as bacteria and archaeal cells, have a cell outer membrane structure that is difficult to effectively lyse; prokaryotic microorganisms have short RNA half-lives and low stability; in addition, the cell size is much smaller than that of mammals cells (10-30 picograms), so the total concentration of RNA molecules in cells is very low, about 4-10 ficks per cell, this RNA concentration is much lower than the RNA concentration in eukaryotic cells, which is RNA Extraction and analysis present many challenges. Although the whole-transcriptome...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2531/119C12Q2525/191C12Q2521/301C12Q2535/122
Inventor 孙诚郭利赵玉琳张建东张连军宋克清吴小彦
Owner 天津迈基生物科技有限公司
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