Plant J. 2010 Jun 1;62(5):898-909. Epub 2010 Mar 4.

Repeat subtraction-mediated sequence capture from a complex genome.

Fu Y, Springer NM, Gerhardt DJ, Ying K, Yeh CT, Wu W, Swanson-Wagner R, D'Ascenzo M, Millard T, Freeberg L, Aoyama N, Kitzman J, Burgess D, Richmond T, Albert TJ, Barbazuk WB, Jeddeloh JA, Schnable PS.

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Department of Agronomy, Iowa State University, Ames, IA 50011, USA.

Abstract

Sequence capture technologies, pioneered in mammalian genomes, enable the resequencing of targeted genomic regions. Most capture protocols require blocking DNA, the production of which in large quantities can prove challenging. A blocker-free, two-stage capture protocol was developed using NimbleGen arrays. The first capture depletes the library of repetitive sequences, while the second enriches for target loci. This strategy was used to resequence non-repetitive portions of an approximately 2.2 Mb chromosomal interval and a set of 43 genes dispersed in the 2.3 Gb maize genome. This approach achieved approximately 1800-3000-fold enrichment and 80-98% coverage of targeted bases. More than 2500 SNPs were identified in target genes. Low rates of false-positive SNP predictions were obtained, even in the presence of captured paralogous sequences. Importantly, it was possible to recover novel sequences from non-reference alleles. The ability to design novel repeat-subtraction and target capture arrays makes this technology accessible in any species.

PMID:: 20230488; [PubMed - indexed for MEDLINE]

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