Improved strategies for sequencing complex genomes using high throughput sequencing technologies

A genome sequence and genome technology, applied in the fields of molecular biology and genetics, can solve problems such as huge computing functions, time-consuming, expensive sequencing methods, etc.

Inactive Publication Date: 2008-10-01
KEYGENE NV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] One of the disadvantages of such short fragments is that the assembly of contigs to determine genome sequence requires enormous computational power, making current sequencing methods relatively expensive and time-consuming

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  • Improved strategies for sequencing complex genomes using high throughput sequencing technologies
  • Improved strategies for sequencing complex genomes using high throughput sequencing technologies
  • Improved strategies for sequencing complex genomes using high throughput sequencing technologies

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Embodiment Construction

[0024] The invention provides a method for detecting genome sequence, which comprises the following steps:

[0025] (a) providing restriction fragments by digesting the genome with at least one first restriction endonuclease to provide a first subset of the genome;

[0026] (b) ligating at least one adapter to the first subset of restriction fragments to provide the first subset of adapter-ligated restriction fragments;

[0027] (c) selectively amplifying a first subset of adapter-ligated restriction fragments using a first primer combination to provide an amplified first subset of adapter-ligated restriction fragments, wherein at least the first primer comprises a primer complementary to the adapter and A fragment complementary to a part of the restriction endonuclease recognition sequence, further comprising a first selected sequence at the 3' end of the primer sequence, wherein the first selected sequence comprises 1-10 selected nucleotides;

[0028](d) repeating step (c) ...

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Abstract

A method for determining a genome sequence comprising the steps of digesting the genome with at least one first restriction endonuclease, ligating at least one adaptor to the restriction fragments of the first subset, selectively amplifying the first set of adaptor-ligated restriction fragments using a first primer combination wherein at least a first primer contains a first selected sequence at the 3' end of the primer sequence, comprising 1-10 selective nucleotides, repeating these steps with at least a second primer combinations wherein the primer contains a different second selected sequence, fragmenting each of the subsets of amplified adaptor-ligated restriction fragments to generate sequencing libraries, determine the nucleotide sequence of the fragments, aligning the sequence of the fragments in each of the libraries to generate contigs, repeating these steps for one second and / or further restriction endonucleases, aligning the contigs obtained for each of the second and / or further restriction endonucleases to provide for a sequence of the genome.

Description

technical field [0001] The present invention relates to molecular biology and genetics. The present invention relates to an improved strategy for the determination of preferably complex (eg large) genomic sequences based on the application of high throughput sequencing technologies. Background of the invention [0002] Assembly of complete genome shotgun sequences of large genomes (above 100Mbp) is a complex problem for mapping genome sequences. Many plants and animals contain even larger numbers of repeats, thus compounding the problem. The advent of high-throughput sequencing technologies such as the 454LifeScience technology further exacerbates this computational challenge. These techniques often no longer rely on Sanger dideoxy sequencing and rely primarily on sequencing-by-synthesis (pyrosequencing), which is easily performed on solid surfaces. Sequencing by synthesis provides a relatively large number of sequences with relatively short sequence lengths (approximatel...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6827C12Q1/6869C12Q2539/107C12Q2531/113C12Q2525/191C12Q2521/301
Inventor M·J·T·范艾克A·P·索兰森R·C·J·霍格斯
Owner KEYGENE NV
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