PT  - JOURNAL ARTICLE
AU  - Satsuki Tsuji
AU  - Masaki Miya
AU  - Masayuki Ushio
AU  - Hirotoshi Sato
AU  - Toshifumi Minamoto
AU  - Hiroki Yamanaka
TI  - Evaluating intraspecific diversity of a fish population using environmental DNA: An approach to distinguish true haplotypes from erroneous sequences
AID  - 10.1101/429993
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 429993
4099  - http://biorxiv.org/content/early/2018/10/15/429993.short
4100  - http://biorxiv.org/content/early/2018/10/15/429993.full
AB  - 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 in HTS data are detected as false positive haplotypes; therefore, reliable strategies are necessary to eliminate them for evaluation of the intraspecific diversity using eDNA analysis. In this study, we propose an approach combining the denoising using amplicon sequence variant (ASV) method and the removal of haplotypes based on detection rates. A mixture of rearing water including nine haplotypes of Ayu (Plecoglossus altivelis altivelis) mitochondrial D-loop region was used as an eDNA sample, and the 15 replicates of sequencing libraries were prepared. All library replications were sequenced by HTS, and the total number of detected true haplotypes and false positive haplotypes were compared with and without the denoising using the ASV method. As a result, the use of the ASV method considerably reduced the number of false positive haplotypes from 5,692 to 31, and it detected 8/9 true haplotypes. In addition, eight true haplotypes were detected in all 15 library replicates; however, false positive haplotypes had various detection rates from 1/15 to 15/15. Thus, by removing haplotypes with lower detection rates than 15/15, the number of false positive haplotypes were more reduced from 31 to seven. The approach proposed in this study successfully eliminated most of false positive haplotypes in the HTS data obtained from eDNA samples, which allowed us to improve the detection accuracy for evaluating intraspecific diversity using eDNA analysis