First of all, the position of each human representative AS variant (RASV) exon mapped on genome was investigated to other species genome by using the human-other species genome alignment, which was made by BLASTZ with parameters 'C=0 H=2000 Y=3400' for primates and 'C=0 H=2000' for other mammals, and whose reciprocal best hit regions were only used to analyze. If the mapped coverage and identity of the overlapped region of the full-length or ORF in exon were more than threshold (Coverage = 70 % and Identity = 60 %), it was identified as "Genome-conserved". If the full-length or ORF region of the exon was also conserved with that of the other species counterpart RASV exon in the genome alignment with greater or equal in the same threshold, it was identified as "Transcript-conserved". If the exon was not mapped on the genome alignment or the mapped coverage and identity were less than the threshold, the exon was identified as "Non-conserved" (Figure 1A).
Figure 1A. Scheme of genomic conservation about human exons compared with other specis.
Using the conservation category of exon level, we determined also the conservation category about transcript level. If at least one Non-conserved exon existed in the transcript, it was identified as "Non-conserved". If the Non-conserved exons did not exist and Genome-conserved exons were included in it, it was determined as "Genome-conserved". If only Transcript-conserved exons were included in it, it was determined as "Transcript-conserved" (Figure 1B).
Figure 1B. Concept of conserved transcript determination about human by using the exon conservation level.
Among Transcript-conserved RASVs, if all exons in not only the human transcript but also the other species counterpart were consisted of Transcript-conserved exons, they were determined as "Equally-spliced variants (ESVs)". Lastly, if two or more ESVs existed in the locus, they were determined as "Conserved AS" (Figure 1C).
Figure 1C. Higher conserved level of Transcript-conserved RASVs.