How are the SIRV isoforms designed?

The SIRV isoform design is based on 7 human model genes. The annotated transcripts of these genes were extended by additional isoforms and variants to comprehensively cover alternative splicing, start- and end-site variations, antisense and overlapping transcripts. The exonic sequences of the resulting 69 transcript structures (6-18 per gene) were derived from database-derived genomes, which were then altered to completely lose alignment identity. Additionally, each sequence was blasted against the NCBI database on both the nucleotide and the protein level to ensure they were non-identical. Intronic sequences were generated randomly.

The SIRV sequences are conform to the canonical exon-intron junction rule: 96.9% of all SIRV junctions are GT-AG, with the less frequent variants being present at 1.7% (GC-AG) and 0.6% (AT-AC). Two non-canonical splice sites were included at 0.4% each (CT-AG and CT-AC).