A MinION™‐based pipeline for fast and cost‐effective DNA barcoding

Abstract

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory and is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrating that one flow cell can generate barcodes for ~500 specimens despite the high basecall error rates of MinION™ reads. The pipeline overcomes these errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. Consensus barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. They are addressed with an optional error correction pipeline that is based on conserved amino acid motifs from publicly available barcodes. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (i) 511 specimens of a mixed Diptera sample, (ii) 575 specimens of ants and (iii) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION™ barcodes for 490 of the 511 specimens which were assessed against reference Sanger barcodes (N = 471). Overall, the MinION™ barcodes have an accuracy of 99.3%-100% with the number of ambiguous bases after correction ranging from <0.01% to 1.5% depending on which correction pipeline is used. We demonstrate that it requires ~2 hr of sequencing to gather all information needed for obtaining reliable barcodes for most specimens (>90%). We estimate that up to 1,000 barcodes can be generated in one flow cell and that the cost per barcode can be <USD 2.

A Sample-to-Report Solution for Taxonomic Identification of Cultured Bacteria in the Clinical Setting Based on Nanopore Sequencing

“…do not yet allow for an analysis at the read level. De novo assembly or consensus generation from individual ONT reads are therefore commonly used to generate sequences that are virtually free from substitution errors (10). Additionally, "polishing" tools can be applied to remove remaining non-random errors such as indels in homopolymer regions from the generated consensus sequences (10)(11)(12)(13).…”

MinION-based DNA barcoding of preserved and non-invasively collected wildlife samples

“…The ability to use field-friendly DNA extraction protocols with these sample types will help to overcome logistical challenges, such as the need for cumbersome or expensive equipment, for wildlife field research. The accuracy of our species identifications is on par with previous MinION DNA barcoding studies and pipelines (Pomerantz et al, 2018;Srivathsan et al, 2018Srivathsan et al, , 2019Krehenwinkel, Pomerantz, Henderson, et al, 2019;Maestri et al, 2019). For all tissue types, extraction methods, and subsets tested with our pipeline, we obtained high quality reads and a consensus sequence that matched >99.29% and at least 419/421 bp to the Sanger sequence for each sample.…”

“…To date, MinION DNA barcoding pipelines have used either assembly (Pomerantz et al, 2018;Krehenwinkel, Pomerantz, Henderson, et al, 2019), clustering-based (Maestri et al, 2019), or alignment (Srivathsan et al, 2018(Srivathsan et al, , 2019 methods. Assembly approaches generally work more consistently for longer barcodes (~1kb), as the underlying software were originally designed for assembling long reads for genome assemblies rather than amplicons.…”

Biodiversity seen through the perspective of insects: 10 simple rules on methodological choices and experimental design for genomic studies

“…The use of MinION™ in DNA barcoding in insects has proven to be fast (ca. 2 hours), cheap (<USD 2 per sample) and reliable when correction pipelines are used to overcome the yet high basecall error rates (> 10%) (Mardis, 2017;Shendure et al, 2017;Srivathsan et al, 2018).…”