Application of genotyping-by-sequencing on semiconductor sequencing platforms: a comparison of genetic and reference-based marker ordering in barley

PLoS One. 2013 Oct 3;8(10):e76925. doi: 10.1371/journal.pone.0076925. eCollection 2013.

Abstract

The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromosome Mapping / instrumentation*
  • Chromosome Mapping / methods
  • Chromosomes, Plant / chemistry*
  • Gene Library
  • Genetic Markers
  • Genome, Plant*
  • Genotype*
  • High-Throughput Nucleotide Sequencing
  • Hordeum / classification
  • Hordeum / genetics*
  • Molecular Sequence Data
  • Molecular Typing / instrumentation*
  • Molecular Typing / methods
  • Polymorphism, Single Nucleotide
  • Semiconductors
  • Sequence Alignment

Substances

  • Genetic Markers

Grant support

This research was supported in part by the United States Department of Agriculture – Agricultural Research Service (Appropriation No. 5430-21000-006-00D) and Kansas State University as well as funds from the German Ministry of Education and Research (BMBF fund TRITEX-0315954A) and EU FP7 project TriticeaeGenome to NS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.