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Genome simplification and next-generation sequencing-based deoxyribose nucleic acid (DNA) library preparation method and kit

A DNA library and next-generation sequencing technology, applied in chemical libraries, biochemical equipment and methods, combinatorial chemistry, etc., can solve problems such as unclear genealogy and low quality

Inactive Publication Date: 2013-01-16
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, for diploid heterozygous species derived from outbreeding populations (Outbreeding popμLation), especially species with unclear pedigrees, low-quality reference sequences, and no haplotype maps, such as pigs, these methods are not fully applicable.

Method used

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  • Genome simplification and next-generation sequencing-based deoxyribose nucleic acid (DNA) library preparation method and kit
  • Genome simplification and next-generation sequencing-based deoxyribose nucleic acid (DNA) library preparation method and kit
  • Genome simplification and next-generation sequencing-based deoxyribose nucleic acid (DNA) library preparation method and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Establishment of DNA library construction method based on genome simplification and next-generation sequencing.

[0055] In this example, heterozygous diploid Landrace and Large white pigs were selected as experimental materials, and the basic operation procedures based on genome simplification and next-generation sequencing DNA library construction are shown in figure 1 , including the following steps:

[0056] The first step, the extraction of genomic DNA: collect pig ear tissue samples, use tissue sample DNA extraction kit to extract genomic DNA, by figure 2 Agarose gel electrophoresis showed that the DNA had no protein and RNA contamination, and the integrity was good. The DNA sample was diluted to ~50ng / μL for use;

[0057] The second step, genomic DNA fragmentation

[0058] The high-quality genomic DNA extracted in the first step was digested (Digestion) with the methylation-sensitive restriction enzyme (Restriction enzyme) AvaII. The recognition sequence of t...

Embodiment 2

[0105] High-throughput porcine genome-wide SNP detection.

[0106] Use genome-based simplification and next-generation sequencing DNA library construction kits for research. Based on genome simplification and next-generation sequencing DNA library construction kit includes:

[0107] Primer1.1 primer and Primer2.1 primer with the following base sequences;

[0108] Primer1.1:

[0109] 5′AATGATACGGCGACCACCGAGATTCACTCTTTCCCTACACGACGCTCTTCCGATCT

[0110] Primer2.1:

[0111] 5′CAAGCAGAAGACGGCATACGAGATCGGTCTCGGCATTCCTGCTGAACCGCTCTTCCGATCT

[0112] PCR phusion mix;

[0113] Restriction endonuclease AvaII and its corresponding buffer;

[0114] T4 DNA ligase;

[0115] 72 pairs of barcode-adapter sequences, each pair of sequence structure is:

[0116] Positive strand: 5'ACACTCTTTCCCTACACGACGCTCTTCCGATCTXXXXX3',

[0117] Negative strand: 5'GWCYYYYYAGATCGGAAGAGCGGTTCAGCAGGAATGCCGAG3',

[0118] XXXXX in the positive chain represents the barcode sequence, YYYYY in the negative chai...

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Abstract

The invention discloses a genome simplification and next-generation sequencing-based deoxyribose nucleic acid (DNA) library preparation method and a kit in the field of biotechnologies. The method and the kit aim to overcome the defects of the conventional library preparation method and can be used for whole genome single nucleotide polymorphism (SNP) detection and genetic typing of species of which the reference genomes are not perfect and the genealogies of research groups are not clear, and which do not have haplotype maps. The DNA library preparation method and the kit are simple in operation process, the prepared library is high in sequencing quality, the segment distribution among individuals is low in variability, the research cost is low, and the DNA library preparation method and the kit have very wide application prospect in the aspect of realizing the high-throughout whole-genome SNP detection and genetic typing research.

Description

technical field [0001] The present invention relates to a method and kit in the field of biotechnology, in particular to a DNA library construction method and kit based on genome simplification and next-generation sequencing. Background technique [0002] With the continuous development of molecular biology, genetics, statistical genomics and other disciplines, methods such as Genome-wide association study (GWAS) are used to study the relationship between human diseases and important agricultural economic traits of livestock at the genome level. Related genetic variation becomes possible, and single nucleotide polymorphism (Single nucleotide polymorphism, SNP) is the basis of GWAS and many research work such as genome selection (Genomic selection), high-precision genetic mapping (Highly-resolution mapping). For SNP detection with fewer sites, such as a few candidate genes (Candidate genes) or a few regions of interest and a few known target SNP sites, many methods such as RT...

Claims

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Application Information

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IPC IPC(8): C40B50/06C12Q1/68
Inventor 潘玉春陈强杨玉梅王起山张向喆马育芳陈振亮廖荣荣涂盈盈颉孝贤王振贺鹏飞张哲
Owner SHANGHAI JIAO TONG UNIV
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