Leibniz Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany.
PLoS One. 2013 Oct 3;8(10):e76925. doi: 10.1371/journal.pone.0076925. eCollection 2013.
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.
下一代测序平台的快速发展使得测序技术能够在一系列遗传学研究和育种应用中用于常规基因分型。测序的基因分型（GBS）是一种低成本、低复杂度的测序方法，正在成为许多物种全基因组标记分析的常用方法。随着测序技术的快速发展，将当前的 GBS 方法应用于新平台将利用这些进展进行未来的研究。为了测试用于 GBS 的新型半导体测序平台，我们对大麦重组自交系（RIL）群体进行了基因分型。基于先前的 GBS 方法，我们为 Ion Torrent 平台设计了条形码和适配器集。构建了四组 24 plex 文库，由 94 个 RIL 和两个亲本组成，并在两个 Ion 平台上进行测序。同时，在 Illumina HiSeq 2000 上对相同的 RIL 96 plex 文库进行了测序。我们应用了两种不同的计算管道来分析测序数据；无参考 TASSEL 管道和基于 SAMtools 的参考管道。用于读取映射和变异调用的序列拼接位于集成的物理和遗传图谱上。我们发现不同平台之间的基因型调用高度一致，遗传标记顺序与参考标记顺序高度一致。然而，不同管道共同发现的 SNP 数量很少，这表明对齐和过滤参数对 SNP 发现有很大的影响。我们展示了当前大麦基因组组装作为开发非常低成本遗传图谱的框架的实用性，促进了高分辨率遗传图谱的构建，并消除了未来在大麦中进行研究时开发从头遗传图谱的需要。通过在半导体测序平台上展示 GBS，我们得出结论，GBS 方法适用于多种平台，并且可以随着新测序技术、分析工具和基因组资源的发展而轻松修改。
