Figure 2

Using Seq4SNPs software as part of the Workflow in SNPstream genotyping sequence preparation. After choosing 48 or more SNPs to assay (START), input to Seq4SNPs software (grey box) is a text file (white, top) containing a list of rs numbers and assay names and the desired assay size (e.g. 200 nucleotides each side of the assay SNP). The first process (blue box, top) outputs a file of sequences in fasta format (white, centre). The user submits this to Repeat Masker which quickly produces a masked fasta file (white, right); at this point the user might reject some assays and start again if insufficient assays remain. In the second process (blue box, bottom) the Seq4SNPs fasta file is automatically used to annotate and reformat. The user inputs the masked file for this step. Final outputs (white, bottom) are a formatted sequence file for assay design software, and a report for Excel, containing warnings, error flags, minisequences for adjacent SNPs and links to dbSNP and is used for assisted error checking. User time is mainly confined to the delay points (green). The formatted assays are submitted to the SNP-IT at Autoprimer (Assay Designer), which rejects sequences that are insufficiently repeat-free, when the user may choose alternatives again (upward arrows). Legend: Seq4SNPs (grey box): times (sec ") are per SNP and are improving. Start and end points (circles); processes (blue rectangles); file/stored output (white rectangles); additional software decision aids (yellow diamonds).