Primer Design for Comparative Genomic Studies

Human reference mRNA sequences for 263 genes were retrieved from the NCBI Reference Sequence Database and individually aligned to their respective Pongo abelli (Sumatran orangutan) genome sequence (NCBI Pongo_pygmaeus_abelii-2.0.2 assembly) via BLAST to find homologous sequence stretches containing as few nucleotide ISVs as possible. The genes in this study were chosen based on our own research interests: the majority of the genes are related to epigenetic regulation mechanisms (Supplementary Table 3). Homologous sequence stretches were subjected to Primer322 (link) to design primer pairs that bind both human and orangutan sequences simultaneously in PCR amplicon while possessing the small numbers of ISV nucleotides in its amplicons to discriminate species origin. Primers were pooled into 24 groups of ~20 pairs in such a way that multiple primer pairs for a single transcript are separately assigned to different groups, to avoid the production of unintended amplicons. A multiplexed PCR reaction was carried out using each primer group and either of two Taq polymerases, Solg^TM h-Taq DNA polymerase (SolGent, Korea) or FastStart Taq polymerase (Roche) for the Rep1 set or Rep2 set, respectively, with the following conditions: 15 min of enzyme activation at 95 °C followed by 40 cycles of 95 °C for 20 s, 55 °C for 25 s, and 65 °C for 2 min.