High-throughput SNP discovery and assay development in common bean

Abstract

BackgroundNext generation sequencing has significantly increased the speed at which single nucleotide polymorphisms (SNPs) can be discovered and subsequently used as molecular markers for research. Unfortunately, for species such as common bean (Phaseolus vulgaris L.) which do not have a whole genome sequence available, the use of next generation sequencing for SNP discovery is much more difficult and costly. To this end we developed a method which couples sequences obtained from the Roche 454-FLX system (454) with the Illumina Genome Analyzer (GA) for high-throughput SNP discovery.ResultsUsing a multi-tier reduced representation library we discovered a total of 3,487 SNPs of which 2,795 contained sufficient flanking genomic sequence for SNP assay development. Using Sanger sequencing to determine the validation rate of these SNPs, we found that 86% are likely to be true SNPs. Furthermore, we designed a GoldenGate assay which contained 1,050 of the 3,487 predicted SNPs. A total of 827 of the 1,050 SNPs produced a working GoldenGate assay (79%).ConclusionsThrough combining two next generation sequencing techniques we have developed a method that allows high-throughput SNP discovery in any diploid organism without the need of a whole genome sequence or the creation of normalized cDNA libraries. The need to only perform one 454 run and one GA sequencer run allows high-throughput SNP discovery with sufficient sequence for assay development to be performed in organisms, such as common bean, which have limited genomic resources.

A reference genome for common bean and genome-wide analysis of dual domestications

“…Linkage maps were constructed using JoinMap 4.0 (ref. 47) software on the basis of the 6,531 polymorphic SNPs from these 2 BeadChips and 484 SNP loci that were genotyped with the Illumina GoldenGate assay at the US Department of Agriculture-Agricultural Research Service in Beltsville, Maryland 48 , as well as 261 SSR markers and 25 framework markers. The final map contained 7,276 SSR and SNP markers arranged in 11 linkage groups via framework markers.…”

Genetic diversity analysis of yellow mustard (Sinapis alba L.) germplasm based on genotyping by sequencing

“…RRLs have been widely applied to reduce the complexity of plant genomes (cf. Gore et al 2009;Maughan et al 2009;Deschamps et al 2010;Hyten et al 2010). They are constructed by means of a restriction enzyme (RE) digest followed by fragmentsize selection, and allow for sampling diverse, but identical, genomic regions from several individuals (Altshuler et al 2000).…”

Next-generation sequencing reveals phylogeographic structure and a species tree for recent bird divergences

“…The other extreme in terms of data quality and quantity was found in the white-crowned sparrow system, in which the length of detected loci had several high peaks (Fig. 2), which could be indicative of restriction digest sites within repetitive DNA (Hyten et al, 2010). Also, coverage was uneven ( Table 3), suggesting that repetitive DNA might have used up many of the reads, exhausting coverage for the remaining loci.…”

“…There are several variations on this basic idea in the literature (Whitelaw et al, 2003;Barbazuk et al, 2005;Van Tassell et al, 2008;Wiedmann et al, 2008;Gompert et al, 2010;Hyten et al, 2010;Williams et al, 2010), most resulting in the generation of SNPs, not full loci, and for a handful of individuals or population-level pools of individuals. Here, we demonstrate the utility for phylogeography of a wet lab method for the rapid and cost-effective generation of reduced representation libraries for NGS (in this case, for the 454 platform, although the basic idea is adaptable to any platform).…”