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

item MASCHER, MARTIN - Leibniz Institute Of Plant Genetics And Crop Plant Research
item WU, SHUANGYE - Kansas State University
item St Amand, Paul
item RICO, ALAIN - Life Technologies Corporation
item SARTORI, ALEXANDER - Life Technologies Corporation
item STEIN, NILS - Leibniz Institute Of Plant Genetics And Crop Plant Research
item Poland, Jesse

Submitted to: PLOS ONE
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2013
Publication Date: 10/3/2013
Publication URL:
Citation: Mascher, M., Wu, S., St Amand, P., Rico, A., Sartori, A., Stein, N., Poland, J.A. 2013. Application of genotyping-by-sequencing on semiconductor sequencing platforms: A comparison of genetic and reference-based marker ordering in barley. PLoS One. DOI: 10.1371/journal.pone.0076925.

Interpretive Summary: Genotyping-by-sequencing is an approach that uses ‘next-generation’ sequencing technology for whole-genome characterization of diverse plant and animal populations for genetics and breeding studies. As new sequencing technology develops, the sequencing output is increasing while costs are decreasing. Therefore, it is important to adapt current methods to new technologies to leverage new developments and maximize output. This will increase the number of samples that can be evaluated while decreasing the per sample cost. In the current study, we modified previous protocols that were used for sequencing on the Illumina HiSeq 2000 to test genotyping-by-sequencing on the Ion Torrent PGM and Ion Torrent Proton. We found that the new Ion Torrent platforms were suitable for GBS and gave consistent results with the established Illumina platform. Overall, this study confirmed that this genotyping-by-sequencing approach can be transferred to new sequencing platforms to take advantage of technology advancements that increase sequencing output.

Technical 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 a range of species. With quickly changing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for larger and more robust genetic studies. We demonstrate the utility of semiconductor sequencing platforms for GBS through genotyping a barley recombinant inbred line population. Based on a previous GBS approach, we designed barcode and adapter sets suitable for the Ion Torrent sequencing chemistry. Four sets of 24-plex libraries were constructed consisting of 94 recombinant inbred lines and the two parents and sequenced on the Ion platforms. In parallel, a 96-plex library of the same lines was sequenced on the Illumina HiSeq 2000. We then implemented a reference-based approach for genetic mapping in barley. Previously anchored reference contigs were used for alignment of sequence tags and single nucleotide polymorphsims were called against this reference sequence. The physically anchored contigs gave a genomic position to each polymorphism and robust genetic maps were constructed against the physical framework. We found high agreement in genotype calls between the different platforms and high concordance between de novo and reference-based genetic maps. We demonstrate 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 amendable to a range of platforms and can easily be modified as new sequencing technologies develop.