Evaluating intraspecific genetic diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences

摘要: Recent advances in environmental DNA (eDNA) analysis using high-throughput sequencing (HTS) provide a non-invasive way to evaluate the intraspecific diversity of aquatic macro-organisms. However, many erroneous sequences included HTS data are detected as false positive haplotypes; therefore, reliable strategies necessary eliminate them for evaluation eDNA analysis. In this study, we propose an approach combining denoising amplicon sequence variant (ASV) method and removal haplotypes based on detection rates. A mixture rearing water including nine Ayu ( Plecoglossus altivelis ) mitochondrial D-loop region was used sample, 15 replicates libraries were prepared. All library replications sequenced by HTS, total number true compared with without ASV method. As result, use considerably reduced from 5,692 31, it 8/9 haplotypes. addition, eight all replicates; however, had various rates 1/15 15/15. Thus, removing lower than 15/15, more 31 seven. The proposed study successfully eliminated most obtained samples, which allowed us improve accuracy evaluating