Double digest RADseq: an inexpensive method for de novo SNP discovery and genotyping in model and non-model species.
The ability to efficiently and accurately determine genotypes is a keystone technology in modern genetics, crucial to studies ranging from clinical diagnostics, to genotype-phenotype association, to reconstruction of ancestry and the detection of selection. To date, high capacity, low cost genotyping has been largely achieved via "SNP chip" microarray-based platforms which require substantial prior knowledge of both genome sequence and variability, and once designed are suitable only for those targeted variable nucleotide sites. This method introduces substantial ascertainment bias and inherently precludes detection of rare or population-specific variants, a major source of information for both population history and genotype-phenotype association. Recent developments in reduced-representation genome sequencing experiments on massively parallel sequencers (commonly referred to as RAD-tag or RADseq) have brought direct sequencing to the problem of population genotyping, but increased cost and procedural and analytical complexity have limited their widespread adoption. Here, we describe a complete laboratory protocol, including a custom combinatorial indexing method, and accompanying software tools to facilitate genotyping across large numbers (hundreds or more) of individuals for a range of markers (hundreds to hundreds of thousands). Our method requires no prior genomic knowledge and achieves per-site and per-individual costs below that of current SNP chip technology, while requiring similar hands-on time investment, comparable amounts of input DNA, and downstream analysis times on the order of hours. Finally, we provide empirical results from the application of this method to both genotyping in a laboratory cross and in wild populations. Because of its flexibility, this modified RADseq approach promises to be applicable to a diversity of biological questions in a wide range of organisms.
Department of Organismic & Evolutionary Biology, Museum of Comparative Zoology, Harvard University, Cambridge, Massachusetts, United States of America. bpeterson@oeb.harvard.edu
Brant K
BK
Peterson
Jesse N
JN
Weber
Emily H
EH
Kay
Heidi S
HS
Fisher
Hopi E
HE
Hoekstra
D000818:Animals; D021461:Animals, Outbred Strains; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D016678:Genome; D005838:Genotype; D020641:Polymorphism, Single Nucleotide; D015203:Reproducibility of Results; D012377:Rodentia; D017422:Sequence Analysis, DNA / Q000379:methods*
D004262:DNA Restriction Enzymes