Using High-Throughput Amplicon Sequencing to Evaluate Intragenomic Variation and Accuracy in Species Identification of Species.

While recent sequencing technologies (third generation sequencing) can successfully sequence all copies of nuclear ribosomal DNA (rDNA) markers present within a genome and offer insights into the intragenomic variation of these markers, high intragenomic variation can be a source of confusion for high-throughput species identification using such technologies. High-throughput (HT) amplicon sequencing via PacBio SEQUEL I was used to evaluate the intragenomic variation of the ITS region and D1-D2 LSU domains in nine species, and the accuracy of such technology to identify these species based on molecular phylogenies was also assessed. PacBio sequences within strains showed variable level of intragenomic variation among the studied species with showing greater variation than the others. Some variants from a mix of species clustered together outside their respective species of origin, indicative of intragenomic variation that escaped concerted evolution shared between species. Proper selection of consensus sequences from HT amplicon sequencing is a challenge for interpretation of correct species identification. PacBio consensus sequences with the highest number of reads represent the major variants within a genome and gave the best results in terms of species identification.