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

<div><p>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 <i>de novo</i> 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.</p> </div

Genotyping-by-sequencing marker order based on the International Barley Sequencing Consortium reference framework (y-axis) compared to a <i>de novo</i> genetic order (x-axis) from 94 Morex x Barke recombinant inbred lines genotyped on either the Ion Torrent PGM or Proton platform.

<p>Each dot corresponds to one of 1,584 markers from the <i>de </i><i>novo</i> map that was positioned to the physical and genetic framework of barley.</p

Relationship between number of sequence reads, missing data, sequencing depth and the number of SNP calls.

<p>(<b>a</b>) The average percentage of missing data per SNP in each sequenced sample is plotted as function of the number of sequence reads in that sample. (<b>b</b>) Histogram of missing data per SNP. (<b>c</b>) The number of SNP calls plotted against the minimum depth at a variant position in a given sample to make a successful genotype call. All SNP calls were made with the SAMtools pipeline. The minor allele frequency was set to 30% and the maximum rate of missing data was set to 50%. The sequencing platforms used for this study include Illumina HiSeq2000 (black), Ion Torrent PGM (green) and Ion Torrent Proton (red). The color code for all panels is given in the legend to (<b>a</b>).</p

Whole genome sequence (WGS; genomic) and transcriptome (RNA) data generated in this study from B73 inbred lines of set A and B plants, respectively.

<p>Whole genome sequence (WGS; genomic) and transcriptome (RNA) data generated in this study from B73 inbred lines of set A and B plants, respectively.</p

Distribution of SNPs and differentially expressed genes along the length of the 10 maize chromosomes (separated by blue lines).

<p>(<b>A</b>) Number in non-overlapping 100 kb windows of genomic SNPs differentiating between plants of set A and B, respectively. (<b>B</b>) Number in non-overlapping 100 kb windows of transcriptome SNPs differentiating between the three replicates from set A and the three replicates from set B. (C) Number in non-overlapping 10 Mb windows of differentially expressed genes (q value ≤0.05, log fold change ≥2).</p

Analysis of putative <i>de novo</i> SNPs and introgression loci in both B73 inbred lines.

<p>(<b>A</b>) Distribution of putative <i>de novo</i> SNPs along the maize genome. <i>De novo</i> SNPs of set A plants are shown to the left of the chromosome ideograms, SNPs of set B plants are shown to the right. The locations of the putative introgressions on chromosomes 1, 5, and 10 in plant B are highlighted in red and drawn to scale. (<b>B</b>) Spectrum of <i>de novo</i> mutations in set A and set B plants, respectively.</p

Phenotypic comparison of progeny plants from two maize B73 inbred lines grown for generations either exclusively in the field (set A) or in the greenhouse (set B).

<p>Plants were grown in compartmented pots with (+AM) or without (−AM) arbuscular mycorrhiza with phosphate addition to the hyphal compartment. (<b>A</b>) Comparison of plant growth phenotype. Pictures have been taken before harvest, approx. 7 weeks after sowing. (<b>B</b>) Comparison of dry weight (DW) and number of green leaves from 3–4 pooled plants of both inbred lines (set A and set B) analyzed in autumn 2010. Significant differences between the treatments are indicated by different letters (n = 3–4, <i>p</i>≤0.05, one-way ANOVA).</p

Introgression intervals in the genome of set B plants of maize inbred line B73.

<p>Introgression intervals in the genome of set B plants of maize inbred line B73.</p

Top20^a differentially expressed genes between B73 inbred lines of set A and B with functional annotation.

<p>Top20^{<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096782#nt103" target="_blank">a</a>} differentially expressed genes between B73 inbred lines of set A and B with functional annotation.</p

Comparison of elemental composition in source leaves and dry seeds of progeny plants from both B73 inbred lines (set A and B).

<p>(<b>A</b>) PCA-analysis of ionomics data from source leaves. Three plants were each grown in compartmented pots with (+AM) or without (−AM) arbuscular mycorrhiza with phosphate addition to the hyphal compartment. (<b>B</b>) PCA-analysis of ionomic data from dry seeds (n = 3). (C) Depiction of 20 elements separately as bar charts. Significant differences between plants of set A and set B are labeled by an asterisk (n = 4–5, student’s t-test, <i>p</i>≤0.05).</p