Repeat subtraction-mediated sequence capture from a complex genome.
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.
Fu
Yan
Y
Department of Agronomy, Iowa State University, Ames, IA 50011, USA.
Springer
Nathan M
NM
Gerhardt
Daniel J
DJ
Ying
Kai
K
Yeh
Cheng-Ting
CT
Wu
Wei
W
Swanson-Wagner
Ruth
R
D'Ascenzo
Mark
M
Millard
Tracy
T
Freeberg
Lindsay
L
Aoyama
Natsuyo
N
Kitzman
Jacob
J
Burgess
Daniel
D
Richmond
Todd
T
Albert
Thomas J
TJ
Barbazuk
W Brad
WB
Jeddeloh
Jeffrey A
JA
Schnable
Patrick S
PS
D055028:Comparative Genomic Hybridization; D018744:DNA, Plant / Q000235:genetics; D017343:Genes, Plant; D018745:Genome, Plant; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D020641:Polymorphism, Single Nucleotide; D017422:Sequence Analysis, DNA / Q000379:methods*; D003313:Zea mays / Q000235:genetics
D018744:DNA, Plant