SNP Discovery and Genotyping for Evolutionary Genetics Using RAD Sequencing

Abstract

Next-generation sequencing technologies are revolutionizing the field of evolutionary biology, opening the possibility for genetic analysis at scales not previously possible. Research in population genetics, quantitative trait mapping, comparative genomics, and phylogeography that was unthinkable even a few years ago is now possible. More importantly, these next-generation sequencing studies can be performed in organisms for which few genomic resources presently exist. To speed this revolution in evolutionary genetics, we have developed Restriction site Associated DNA (RAD) genotyping, a method that uses Illumina next-generation sequencing to simultaneously discover and score tens to hundreds of thousands of single-nucleotide polymorphism (SNP) markers in hundreds of individuals for minimal investment of resources. In this chapter, we describe the core RAD-seq protocol, which can be modified to suit a diversity of evolutionary genetic questions. In addition, we discuss bioinformatic considerations that arise from unique aspects of next-generation sequencing data as compared to traditional marker-based approaches, and we outline some general analytical approaches for RAD-seq and similar data. Despite considerable progress, the development of analytical tools remains in its infancy, and further work is needed to fully quantify sampling variance and biases in these data types.

RAD-seq derived genome-wide nuclear markers resolve the phylogeny of tunas

“…Restriction-site-associated DNA libraries were prepared following the methods of Etter et al (2011). Briefly, about 300 ng of genomic DNA were digested with the SbfI restriction enzyme and ligated to modified Illumina P1 adapters containing 5bp unique barcodes.…”

Genomic tools for new insights to variation, adaptation, and evolution in the salmonid fishes: a perspective for charr

“…Reviews and protocols outlining how genotypes are inferred from NGS sequences in detail can be found elsewhere Etter et al, 2011;Catchen et al, 2013;Recknagel et al, 2015) and therefore will not be covered in detail here. Briefly, the read is sequenced from the restriction enzyme cut site, either in one direction (single-end sequencing) or from two directions (paired end sequencing).…”

Large-scale genotyping of highly polymorphic loci by next-generation sequencing: how to overcome the challenges to reliably genotype individuals?

“…Then, the parameters of the model proposed by Galan et al (2010) could be changed according to the determined number of sequenced loci or using an extension of this model proposed by Sommer et al (2013), that allows to take into account variation in amplification efficiency, a common pattern when amplifying several loci with a unique pair of primers. Additionally, a modification of the maximum-likelihood framework proposed by Hohenlohe et al (2010) and Etter et al (2011) is required to determine homozygous and heterozygous amplicons for recently duplicated genes.…”

“…Determination of homozygous and heterozygous amplicons. To determine homozygous and heterozygous amplicons, the model proposed by Hohenlohe et al (2010), and later used by Etter et al (2011), is used to calculate, given the sequencing error rate, the likelihood of each possible genotype given all the retained variants of an amplicon. An homozygous or an heterozygous genotype is assigned to each amplicon based on a likelihood ratio test between the most likely homozygous and the most likely heterozygous genotypes with one degree of freedom.…”

“…The sequencing error rate for 454 FLX sequencing instrument has been estimated to range between 0.18% and 0.03% (Huse et al, 2007). Sequencing error rate was assumed to be constant at each locus (but see Hohenlohe et al, 2010;Etter et al, 2011). Indeed, at this stage of the post-processing most of the sequencing errors should have been eliminated thanks to the previous post-processing steps (for example, the elimination of singletons and reads of o95% and 4105% correct length variants or the use of indels).…”