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Methods and systems for solution based sequence enrichment

a technology of sequence enrichment and solution, applied in the field of methods and systems for the capture and enrichment of target nucleic acids and analysis, can solve the problems of reducing arduous and often fruitless task of identifying such snps, and using genomes to reduce the complexity of the genom

Inactive Publication Date: 2009-04-23
ROCHE NIMBLEGEN
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  • Abstract
  • Description
  • Claims
  • Application Information

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Benefits of technology

[0009]The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format. Methods and systems of the present invention are useful in aiding investigators and clinicians in identifying, studying and following treatment regimens associated with disease and disease states.
[0011]The disclosed methods provide a cost-effective, flexible and efficient approach for reducing the complexity of a genomic sample. Genomic samples are used herein for descriptive purposes, but it is understood that other non-genomic samples could be subjected to the same procedures. The methods and systems described herein provide for enrichment of target sequences in a solution based approach thereby providing an alternative to microarray substrate based methods for use in research and therapeutics associated with disease and disease states such as cancers (Durkin et al., 2008, Proc. Natl. Acad. Sci. 105:246-251; Natrajan et al., 2007, Genes, Chr. And Cancer 46:607-615; Kim et al., 2006, Cell 125:1269-1281; Stallings et al., 2006 Can. Res. 66:3673-3680), genetic disorders (Balciuniene et al., Am. J. Hum. Genet. In press), mental diseases (Walsh et al., 2008, Science 320:539-543; Roohi et al., 2008, J. Med. Genet. Epub 18 Mar. 2008; Sharp et al., 2008, Nat. Genet. 40:322-328; Kumar et al., 2008, Hum. Mol. Genet. 17:628-638;) and evolutionary and basic research (Lee et al., 2008, Hum. Mol. Gen. 17:1127-1136; Jones et al., 2007, BMC Genomics 8:402; Egan et al., 2007, Nat. Genet. 39:1384-1389; Levy et al., 2007, PLoS Biol. 5:e254; Ballif et al., 2007, Nat. Genet. 39:1071-1073; Scherer et al., 2007, Nat. Genet. S7-S15; Feuk et al., 2006, Nat. Rev. Genet. 7:85-97), to name a few.

Problems solved by technology

Identifying such SNPs has proved to be an arduous and frequently fruitless task because resequencing large regions of genomic DNA, usually greater than 100 kilobases (Kb), from affected individuals or tissue samples is required to find a single base change or to identify all sequence variants.
The genome is typically too complex to be studied as a whole, and techniques must be used to reduce the complexity of the genome.
However, existing methods are limited by, for example, their ease of use and inflexibility of materials and methods.

Method used

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  • Methods and systems for solution based sequence enrichment
  • Methods and systems for solution based sequence enrichment
  • Methods and systems for solution based sequence enrichment

Examples

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example 1

Discovery of New Polymorphisms and Mutations in Large Genomic Regions

[0083]This generic example describes how to perform selection that allows for rapid and efficient discovery of new polymorphisms and mutations in large genomic regions. Microarrays having immobilized probes are used in one- or multiple rounds of hybridization selection with a target of total genomic DNA, and the selected sequences are amplified by LM-PCR

a) Preparation of the Genomic DNA and Double-Stranded Linkers

[0084]DNA is fragmented using sonication to an average size of 500 base pairs. A reaction to polish the ends of the sonicated DNA fragments is set up:

DNA fragments41 μlT4 DNA Polymerase20 μlT4 DNA polymerase reaction mix20 μlWater10 μl

[0085]The reaction is incubated at 11° C. for 30 min. The reaction is then subjected to phenol / chloroform extraction procedures and the DNA is recovered by ethanol precipitation. The precipitated pellet is dissolved in 10 μl water (to give a final concentration of 2 μg / μl).

[0...

example 2

Array-Targeted Resequencing

[0098]A series of high-density oligonucleotide microarrays that capture short segments that correspond to 6,726 individual gene exon regions of at least 500 base pairs were chosen from 660 genes distributed about the human genome (sequence build HG17) (approximately 5 Mb of total sequence) were synthesized according to standard Roche NimbleGen, Inc. microarray manufacturing protocols. Overlapping microarray probes of more than 60 bases each on the array spanned each target genome region, with a probe positioned each 10 bases for the forward strand of the genome.

[0099]Highly-repetitive genomic regions were excluded by design from the capture microarrays, to reduce the likelihood of non-specific binding between the microarrays and genomic nucleic acid molecules. The strategy for identifying and excluding highly-repetitive genomic regions was similar to that of the WindowMasker program (Morgulis et al.). The average 15-mer frequency of each probe was calculat...

example 3

Sequence Variation Captured by Genomic Enrichment and Resequencing

[0106]To ascertain the ability to discern variation in the human genome, genomic DNA samples from four cell types in the human HapMap collection (CEPH / NA11839, CHB / NA18573, JPT / NA18942, YR1 / NA18861, Coriell) were captured on the exon arrays of the prior examples, eluted and sequenced, as disclosed herein, except that the genomic DNAs were not whole genome amplified before capture. The capture results (shown in Table 1, rows 4-7) were similar to those above, except that sequence coverage was consistently more uniform than before, suggesting a bias introduced during WGA.

[0107]The sequence from the four HapMap samples was assembled and mutations were identified and compared to the HapMap SNP data for each sample (Tables 1 and 2). The total number of positions in the target regions that were genotyped in the HapMap project was 8103 (CEU), 8134 (CHB), 8134 (JPT), 8071 (YR1) for each of the four genomes. Of these, most (˜60...

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Abstract

The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format.

Description

[0001]The present application claims priority to European Patent Application Serial Number 07020660.2 filed Oct. 23, 2007, which is incorporated herein by reference in its entirety.FIELD OF THE INVENTION[0002]The present invention provides methods and systems for the capture and enrichment of target nucleic acids and analysis of the enriched target nucleic acids. In particular, the present invention provides for the enrichment of targeted sequences in a solution based format.BACKGROUND OF THE INVENTION[0003]The advent of nucleic acid microarray technology makes it possible to build an array of millions of nucleic acid sequences in a very small area, for example on a microscope slide (e.g., U.S. Pat. Nos. 6,375,903 and 5,143,854). Initially, such arrays were created by spotting pre-synthesized DNA sequences onto slides. However, the construction of maskless array synthesizers (MAS) as described in U.S. Pat. No. 6,375,903 now allows for the in situ synthesis of oligonucleotide sequenc...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B10/00C40B60/12
CPCC12Q1/6813C12Q1/6827C12Q2565/501C12Q2535/138C12Q1/6806C12N15/1006C12Q2537/159C12Q1/6844
Inventor RODESCH, MATTHEWALBERT, THOMAS
Owner ROCHE NIMBLEGEN
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