Omitting end preparation reduces index misassignment in Nanopore-based DNA metabarcoding: application to a decadal coastal time series in the Sea of Okhotsk
- 1Kitasato University; The University of Tokyo
Abstract
Environmental DNA (eDNA) metabarcoding enables sensitive, non-invasive assessment of fish communities, but highly multiplexed analyses using Oxford Nanopore Technologies (ONT) platforms require stringent control of sample-index misassignment and sequencing errors. We developed a MiFish experimental workflow combining unique dual indexes, a library protocol that omitted end preparation and used 5'-phosphorylated primers, BLAST-based demultiplexing, quality-dependent clustering, consensus generation, and haplotype partitioning by SNP/INDEL patterns. Omitting pooled end-prep reduced the mean index-chimera rate from 0.0674% to 0.000420%, a 160-fold reduction. We applied the workflow to an archive of seawater samples collected weekly off Monbetsu, Hokkaido, Japan, from 2012 to 2022. Relative read abundance (RRA) data were obtained for 274 samples, and eight taxa showed significant seasonality. For six of these taxa, the three-year mean RRA peaks coincided with reported spawning periods in Hokkaido. The workflow substantially reduced index misassignment and enabled cost-efficient, highly multiplexed analysis of a decadal fish eDNA time series.
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BibTeX
@misc{endo2026omitting,
title = {{Omitting end preparation reduces index misassignment in Nanopore-based DNA metabarcoding: application to a decadal coastal time series in the Sea of Okhotsk}},
author = {M. Endo and S. Nagai and T. Watanabe and N. Kurita and S. Asakawa and K. Yoshitake},
howpublished = {bioRxiv},
year = {2026},
month = sep,
doi = {10.64898/2026.09.08.750005},
url = {https://www.biorxiv.org/content/10.64898/2026.09.08.750005v1},
note = {CC BY-NC},
}
RIS
TY - UNPB
TI - Omitting end preparation reduces index misassignment in Nanopore-based DNA metabarcoding: application to a decadal coastal time series in the Sea of Okhotsk
AU - M. Endo
AU - S. Nagai
AU - T. Watanabe
AU - N. Kurita
AU - S. Asakawa
AU - K. Yoshitake
PY - 2026
DA - 2026/09/08
PB - bioRxiv
DO - 10.64898/2026.09.08.750005
UR - https://www.biorxiv.org/content/10.64898/2026.09.08.750005v1
AB - Environmental DNA (eDNA) metabarcoding enables sensitive, non-invasive assessment of fish communities, but highly multiplexed analyses using Oxford Nanopore Technologies (ONT) platforms require stringent control of sample-index misassignment and sequencing errors. We developed a MiFish experimental workflow combining unique dual indexes, a library protocol that omitted end preparation and used 5'-phosphorylated primers, BLAST-based demultiplexing, quality-dependent clustering, consensus generation, and haplotype partitioning by SNP/INDEL patterns. Omitting pooled end-prep reduced the mean index-chimera rate from 0.0674% to 0.000420%, a 160-fold reduction. We applied the workflow to an archive of seawater samples collected weekly off Monbetsu, Hokkaido, Japan, from 2012 to 2022. Relative read abundance (RRA) data were obtained for 274 samples, and eight taxa showed significant seasonality. For six of these taxa, the three-year mean RRA peaks coincided with reported spawning periods in Hokkaido. The workflow substantially reduced index misassignment and enabled cost-efficient, highly multiplexed analysis of a decadal fish eDNA time series.
ER -