SNP Filtering and Annotation for Wheat Genotyping

For assay design, SNPs were filtered to remove those that (i) had sequences showing similarity to the repeats (e-value ≤1e−10) identified by comparing 100 bp SNP-flanking sequences with the GIRI (http://www.girinst.org/repbase/) and ITMI Triticeae Repeat Sequence databases (wheat.pw.usda.gov/ITMI/Repeats) and (ii) were located in close proximity (<50 bp) to the exon–intron junctions identified in the wheat genome assembly (Brenchley et al., 2012 (link)). The selected SNPs were then submitted to the Illumina Assay Design Tool for design score calculation (www.illumina.com). A total of 91 829 SNPs were included into the assay design (Table S5).
Synonymous or nonsynonymous SNPs were annotated by comparing sequences with the nonredundant protein database at NCBI (https://www.ncbi.nlm.nih.gov/) using the blastx program with the e-value threshold of ≤1e⁻¹⁰. For functional annotation, RTs were translated into six reading frames and compared against the protein sequences (blastx e-value threshold ≤1e⁻⁰⁵) predicted in the rice, sorghum, maize and barley genomes. The output of the blastx program was used for automated functional annotation using blast2GO (http://www.blast2go.de/).