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Simplified representative unicellular whole-genome database creating method and application thereof

A whole-genome, single-cell technology, applied in biology and simplified genome field, which can solve problems such as base errors, SNV false positives, single nucleotide variations, etc.

Inactive Publication Date: 2018-06-12
SHANGHAI MAJORBIO BIO PHARM TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These mutated uracils, if not repaired, would be amplified in exponential amplification and eventually lead to SNV false positives
In actual operation, cleavage method, fidelity of DNA polymerase, GC content of the amplified region, etc. will lead to base errors, resulting in non-true single nucleotide variation, that is, SNV false positive problem

Method used

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  • Simplified representative unicellular whole-genome database creating method and application thereof
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  • Simplified representative unicellular whole-genome database creating method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0126] Simplified Genomic Library Construction of Human Peripheral Blood Samples

[0127] 1. Take a peripheral blood sample and divide the separated white blood cells into 6 tubes, with 1 cell in each tube to ensure that the same human single cell sample is obtained. Divided into two groups, A and B, respectively named A1 / A2 / A3, B1 / B2 / B3. Group A was amplified according to the above-mentioned technical scheme except that the enzyme treatment step in step 2 was not added, and group B was amplified and library-built completely according to the above-mentioned technical scheme. Take 500 microliters of blood from the same tube for whole blood DNA extraction, named as bulk, for subsequent false positive comparison.

[0128] 2. After the above-mentioned amplified product is purified by magnetic beads, the sequencing depth is 100 times. The bulk samples were simultaneously constructed and sequenced.

[0129] 3. Data analysis: Statistics of the base mutation types and ratios betwee...

Embodiment 2

[0143] To study the genomic differences between catfish single fertilized egg samples in two different regions (region A, region B), the amplification of a single fertilized egg sample and library construction were carried out according to the process in the technical plan, and a single fertilized egg sample of area C perch was used to carry out Conventional genomic library construction, as a control analysis sample. The results of the association analysis are as follows. For the FST analysis of the distribution of the genetic parameters of each population on the genome, please refer to Figure 4 As shown, for the PCA analysis on it, please refer to Figure 5 shown.

[0144] according to Figure 4 , 5 The specific analysis is as follows: FST means F-statistics (F analysis) in genetic analysis. Fst is a measure of genetic differentiation between subpopulations. The extent of genetic relationships between different populations can be quantified. FST analysis found that alt...

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Abstract

The invention provides a better and suitable simplified representative unicellular whole-genome database creating method and application thereof. On one hand, the method can be effectively applied totrace samples or samples with smaller cell population to perform simplified genome database creation by optimizing primers and amplifying steps; on the other hand, by means of enzyme correction treatment, an enzyme treating step capable of specifically resecting uridine monophosphate can be applied to a simplified genome unicellular sample amplifying method, and amplification error rate can be remarkably reduced. Compared with the prior art, the method creatively applies an optimized and improved unicellular database creating scheme to simplified genome database creation of the trace samples or the samples with the smaller cell population; meanwhile, compared with the prior art, the method has the advantages of higher efficiency, simpleness, practicability, accuracy, small loss, low cost,good method repeatability, low error rate, great suitability for the simplified genome unicellular whole-genome amplification of the trace samples or the samples with the smaller cell population and ability in widening sample application ranges; meanwhile, the error rate is reduced, and detection accuracy is improved.

Description

technical field [0001] This patent relates to the fields of biology and simplified genomes, in particular to a simplified representative single-cell genome-wide library method and its application. Background technique [0002] Reduced-representation genome sequencing (RRGS) refers to the use of bioinformatics methods to design marker development programs, enrich specific length fragments, and apply high-throughput sequencing technology to obtain massive tag sequences to sequence part of the genome , represents the sequencing method for the whole genome information of the target species. Simplified genomes are often applied in the analysis of samples without reference genome sequences. The study of population evolution involves a large sample size. Simplified genomes can greatly reduce the cost of sequencing. In addition, using the simplified genome sequencing strategy to study population evolution is not limited by the reference genome, and the range of species studied is w...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/6806C12Q1/6869C40B50/06
CPCC12N15/1093C12Q1/6806C12Q1/6869C40B50/06C12Q2525/191C12Q2521/531C12Q2521/319C12Q2531/113C12Q2535/122
Inventor 陈昌岳李明明李静张璐璐查洋王雨倩胡秋萍张祥林
Owner SHANGHAI MAJORBIO BIO PHARM TECH
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