eDNA haplotyping of an endangered fish Gnathopogon caerulescens

Genetic diversity of an endangered fish Gnathopogon caerulescens was evaluated using environmental DNA (eDNA) metabarcoding, targeting a partial sequence of the mitochondrial cytochrome b (CytB) gene. We proposed an internal standard approach which accurately eliminates erroneous sequencing reads without affecting the sensitivity for low-abundant true sequences. First, we investigated the effect of the number of PCR cycles (30 or 35) employed in sequencing library preparation on the error profiles of the high-throughput sequencing (HTS) data. The mixtures of artificial DNAs of G. caerulescens CytB sequences were used as templates for library preparation and analyzed by HTS. Based on the error profiles, we determined the optimal library preparation conditions and developed the denoising procedures. Second, to investigate the accuracy of the developed approach, mock populations that were prepared using ten artificial DNAs of G. caerulescens CytB haplotypes were analyzed by HTS along with the internal standards. Finally, we carried out eDNA surveys around Lake Biwa and inferred their population genetic structures.