Paired-end library construction method and method for sequencing genome by using library

A library construction, paired-end technology, applied in the field of paired paired-end library construction, can solve problems such as low recovery rate, difficult cyclization, and influence

Inactive Publication Date: 2011-09-14
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Now different sequencing companies have launched different Paired-end library preparation methods, such as Roche, Illumina, and are widely used, but the span of their Paired-end library is within 20kb, mainly because the existing preparation methods first need to The extracted genomic DNA is interrupted to a certain size with HydrogenShear. The larger the fragment length, the lower the recovery rate, resulting i

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  • Paired-end library construction method and method for sequencing genome by using library
  • Paired-end library construction method and method for sequencing genome by using library
  • Paired-end library construction method and method for sequencing genome by using library

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Embodiment 1

[0027] Such as figure 2 As shown, in another embodiment of the present invention, a flowchart of paired paired-end library construction, the specific process is:

[0028] (1) Plasmid DNA extraction: extract large fragments of plasmid DNA;

[0029] (2) Plasmid fragmentation: take no less than 20ug of the plasmid in step (1), shear the sample with an instrument, and obtain a fragment that is 100-1500bp larger than the vector used in the genome library by the method of gel cutting and recovery;

[0030] (3) EcoR I restriction site methylation: under the action of EcoR I methylase, the EcoR I restriction site on the fragment is methylated and protected;

[0031] (4) Fill in the ends of fragments: Fill in the ends of the fragments under the action of T4 DNA polymerase and T4 polynucleotide kinase; connect Hairpin Adaptor: under the action of ligase, add at both ends of the methylated fragments On the Hairpin Adaptor linker, the linker has a stem-loop structure and contains an EcoR I restr...

Embodiment 2

[0039] A paired-end (Paired-End) high-throughput sequencing of a Bacterial Artificial Chromosome (BAC) library with an average insert length of ~80kb.

[0040] 1) Bacchus leucocephalus BAC library: The BAC library used in this experiment is the BAC library of leucocephalus Chinese leucocephalus. The vector of the library is CopyControl? pCC1BAC? Vector (Epicentre), with a total length of 8128bp. The library consists of 44,706 clones, and the average length of the insert is about 80kb.

[0041] 2) Reagents and instruments: The HydroShear genomic DNA shearing instrument from Digilab Genomic Solutions is used for DNA fragmentation. SAM, EcoR I methylase, λ-exonuclease, T7 exonuclease, and exonuclease I used in the experiment were purchased from NEB; bovine serum albumin (BSA), ATP, PCR nucleic acid mixture, T4 DNA polymerase , T4 Polynucleotide Kinase (PNK), Hairpin Adaptor, Rapid Ligase, EcoRI (high concentration), GC-RICH PCR system, GS FLX Titanium Amplicon emPCR kit purchased fro...

Embodiment 3

[0113] A paired-end (Paired-End) high-throughput sequencing of a Bacterial Artificial Chromosome (BAC) library with an average insert length of ~160kb

[0114] 1) Bacchus leucocephalus BAC library: The BAC library used in this experiment is the Bacillus leucocephalus BAC library of China. The vector of the library is CopyControl® pCC1BAC™ vector (Epicentre), with a total length of 8128bp. The library consists of 15,302 clones, with an average insert length of ~160kb.

[0115] 2) Reagents and instruments: The HydroShear Genomic DNA Shearing Machine from Digilab Genomic Solutions is used for DNA fragmentation. SAM, EcoR I methylase, λ-exonuclease, T7 exonuclease, and exonuclease I used in the experiment were all purchased from NEB; bovine serum albumin (BSA), ATP, PCR nucleic acid mixture, T4 DNA polymerase , T4 Polynucleotide Kinase (PNK), Hairpin Adaptor, Rapid Ligase, EcoRI (high concentration), GC-RICH PCR system, GS FLX Titanium Amplicon emPCR kit purchased from Roche; Advantag...

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Abstract

The invention relates to a paired-end library construction method and a method for sequencing a genome by using the library. The paired-end library construction method comprises the following steps of: performing plasmid extraction on clone in a constructed genome DNA library, fragmenting the extracted plasmid DNA, performing methyl protection on the restriction endonuclease site on the fragmented DNA, adding a hairclip type joint, performing enzyme cutting and cyclization to obtain circular DNA, then amplifying the genome library paired-end fragment in the circular DNA through a pair of composite primers to obtain a paired-end sequence of ultra-long span, and finally performing high flux sequencing. The paired-end sequence obtained by using the method can be applied to splicing of a new species genome sequence so as to further improve the splicing quality.

Description

Technical field [0001] The invention relates to a method for constructing a paired paired-end library and a method for genome sequencing using the library. Background technique [0002] The establishment and use of gene libraries is a development of recombinant DNA technology in the early 1970s. In order to isolate genes, especially those of eukaryotes, since 1974, organisms such as Escherichia coli, yeast, fruit flies, chickens, rabbits, mice, humans, soybeans and other organisms have been established successively, as well as the mitochondrial and chloroplast DNA of some organisms. Gene library. The establishment of gene library has brought the research of molecular genetics and genetic engineering into a new era. [0003] After the genomic DNA of an organism is partially digested with restriction enzymes, the digested fragments are inserted into the vector DNA molecules. The collection of all these vector molecules inserted with genomic DNA fragments will contain the entire gen...

Claims

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Application Information

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IPC IPC(8): C40B50/06C40B40/08C12N15/63C12N15/10C12Q1/68
Inventor 徐安龙付永贵周思思
Owner SUN YAT SEN UNIV
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