A workflow for accurate metabarcoding using nanopore MinION sequencing

Abstract

Metabarcoding has become a common approach to the rapid identification of the species composition in a mixed sample. The majority of studies use established short‐read high‐throughput sequencing platforms. The Oxford Nanopore MinIONTM, a portable sequencing platform, represents a low‐cost alternative allowing researchers to generate sequence data in the field. However, a major drawback is the high raw read error rate that can range from 10% to 22%. To test whether the MinIONTM represents a viable alternative to other sequencing platforms, we used rolling circle amplification (RCA) to generate full‐length consensus DNA barcodes for a bulk mock sample of 50 aquatic invertebrate species with at least 15% genetic distance to each other. By applying two different laboratory protocols, we generated two MinIONTM runs that were used to build error‐corrected consensus sequences. A newly developed Python pipeline, ASHURE, was used for data processing, consensus building, clustering and taxonomic assignment of the resulting reads. Our pipeline achieved median accuracies of up to 99.3% for long concatemeric reads (>45 barcodes) and successfully identified all 50 species in the mock community. The use of RCA was integral for increasing consensus accuracy but was also the most time‐consuming step of the laboratory workflow. Most concatemeric reads were skewed towards a shorter read length range with a median read length of up to 1,262 bp. Our study demonstrates that Nanopore sequencing can be used for metabarcoding, but exploration of other isothermal amplification procedures to improve consensus accuracy is recommended.

Haplotype-level metabarcoding of freshwater macroinvertebrate species: a prospective tool for population genetic analysis

“…Hence, the inability of shorter marker regions to differentiate haplotypes of certain species requires further development and evaluation. On the other hand, the advancements in sequencing technologies to generate longer sequences could allow the full-length generation of longer DNA barcodes from community samples (Baloğlu et al, 2021), resolving the limitations of short amplicon sequencing in DNA metabarcoding.…”

“…However, given the high raw read error rate from these long-read sequencing platforms that range from 10-22% (i.e., the Oxford Nanopore MinION TM ), further assessment and exploration of library preparation and error-correction methods are recommended (Baloğlu et al, 2021) for its utilization for DNA metabarcoding studies, more so for haplotype-level inference.…”

A practical guide to DNA-based methods for biodiversity assessment

“…While the above text focuses on the use of Illumina platforms for metabarcoding, Oxford Nanopore Technology devices (particularly the MinION) have also been used in several recent metabarcoding studies to detect target species of toxic microalgae (Hatfield et al 2020), bivalve invasive species ) and even sharks (Truelove et al 2019) and macroinvertebrate communities (Baloğlu et al 2021).…”

DNA Metabarcoding Methods for the Study of Marine Benthic Meiofauna: A Review

“…Sequencing techniques such as PacBio and Nanopore allow sequencing of longer amplicons (Callahan et al, 2019) and have been successfully used for metabarcoding of environmental samples (Heeger et al, 2018;Karst et al, 2018;Davidov et al, 2020;Okazaki et al, 2021). Even though trade-offs regarding the higher error rate and read coverage compared to Illumina short-read sequencing exist, longer reads comprising several genes may provide better taxonomic resolution due to the high informational content per read (Weirather et al, 2017;Baloglu et al, 2021). To our knowledge, these sequencing platforms have not been used in marine meiofaunal metabarcoding studies and might thus be valuable options for future studies (Hebert et al, 2018).…”