Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

Martin Mascher,Nils Stein,Jesse Poland,Shuangye Wu,Paul St Amand,Hector Candela

doi:10.1371/journal.pone.0076925

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.

Highlights

Rapid advancements in DNA sequencing technologies and platforms are lowering sequencing cost while increasing output
We genotyped a set of 94 recombinant inbred lines (RILs) from a cross between barley cultivars ‘Morex’ and ‘Barke’ that were previously used for high-throughput SNP genotyping to genetically anchor the physical map of barley [36,37]
We developed a total of four libraries comprised of Morex and Barke barley cultivars and 94 unique RILs from these two parents for testing GBS on semiconductor sequencing platforms

Summary

Introduction

Rapid advancements in DNA sequencing technologies and platforms are lowering sequencing cost while increasing output. This is bringing routine sequencing of whole genomes within reach [1,2,3,4,5,6]. For larger and more complex genomes, sequencing approaches targeting a reduced representation of the full genome space are needed to reproducibly sequence the same regions across many samples [7]. For many genetic studies, a limited number of markers is sufficient and per sample cost considerations are important for assaying large populations. Robust and low-cost approaches for whole-genome characterization are needed to assay these large populations and collections

Methods

Results

Conclusion