The advantage of rhAmpSeq genotyping platform for highly diverse and heterozygous species 

Previously, we found that the AmpSeq genotyping platform outperforms GBS for highly diverse and heterozygous species, due to limited missing data, increased coverage and accuracy at heterozygote sites, and elevated transferability among species. Different from SNP arrays or KASP, which typically target two alleles per marker, or site, the AmpSeq genotyping platform allows identification of numerous, novel alleles as a short haploblock because the entire amplified region (typically 200bp to 250bp long) is sequenced through NGS. In this study, #% of polymorphic markers had 3 or 4 alleles out of four possible (Table 1). This high information content, even coverage, and unbiased sequencing of amplicons make this platform is applicable in population genetics and ecology studies. Relative to AmpSeq, the rhAmpSeq technology simply adds an RNA base and block DNA at the 3’ end of each primer. When the match is perfect between the primers and template, this RNA-base and blocker are cleaved by RNase H2 enzyme 32. This step increases the genotyping specificity and increases the multiplexing capacity to 5000 markers per reaction (rhAmpSeq, IDT).