Primed and ready: nanopore metabarcoding can now recover highly accurate consensus barcodes that are generally indel-free

DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which ar...

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Published in:	BMC genomics Vol. 25; no. 1; pp. 842 - 16
Main Authors:	Chang, Jia Jin Marc, Ip, Yin Cheong Aden, Neo, Wan Lin, Mowe, Maxine A D, Jaafar, Zeehan, Huang, Danwei
Format:	Journal Article
Language:	English
Published:	England BioMed Central Ltd 09-09-2024 BioMed Central BMC
Subjects:	Accuracy Analysis Animals Bar codes Biodiversity Biological diversity Biological diversity conservation Biomonitoring Control Cytochrome oxidase Cytochrome-c oxidase Deoxyribonucleic acid DNA DNA barcoding DNA Barcoding, Taxonomic - methods DNA sequencing Electron Transport Complex IV - genetics Error correction Error correction & detection Gene sequencing Genetic aspects High-Throughput Nucleotide Sequencing - methods Identification and classification Illumina INDEL Mutation Methods MinION Nanopore Sequencing - methods Nanopores Next-generation sequencing Nucleotide sequence Nucleotide sequencing Plankton Platforms Real time Sequence Analysis, DNA - methods Species diversity Zooplankton Zooplankton - classification Zooplankton - genetics Singapore Species diversity MinION Biomonitoring Next-generation sequencing Illumina Zooplankton
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Summary:	DNA metabarcoding applies high-throughput sequencing approaches to generate numerous DNA barcodes from mixed sample pools for mass species identification and community characterisation. To date, however, most metabarcoding studies employ second-generation sequencing platforms like Illumina, which are limited by short read lengths and longer turnaround times. While third-generation platforms such as the MinION (Oxford Nanopore Technologies) can sequence longer reads and even in real-time, application of these platforms for metabarcoding has remained limited possibly due to the relatively high read error rates as well as the paucity of specialised software for processing such reads. We show that this is no longer the case by performing nanopore-based, cytochrome c oxidase subunit I (COI) metabarcoding on 34 zooplankton bulk samples, and benchmarking the results against conventional Illumina MiSeq sequencing. Nanopore R10.3 sequencing chemistry and super accurate (SUP) basecalling model reduced raw read error rates to ~ 4%, and consensus calling with amplicon_sorter (without further error correction) generated metabarcodes that were ≤ 1% erroneous. Although Illumina recovered a higher number of molecular operational taxonomic units (MOTUs) than nanopore sequencing (589 vs. 471), we found no significant differences in the zooplankton communities inferred between the sequencing platforms. Importantly, 406 of 444 (91.4%) shared MOTUs between Illumina and nanopore were also found to be free of indel errors, and 85% of the zooplankton richness could be recovered after just 12-15 h of sequencing. Our results demonstrate that nanopore sequencing can generate metabarcodes with Illumina-like accuracy, and we are the first study to show that nanopore metabarcodes are almost always indel-free. We also show that nanopore metabarcoding is viable for characterising species-rich communities rapidly, and that the same ecological conclusions can be obtained regardless of the sequencing platform used. Collectively, our study inspires confidence in nanopore sequencing and paves the way for greater utilisation of nanopore technology in various metabarcoding applications.
Bibliography:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1471-2164 1471-2164
DOI:	10.1186/s12864-024-10767-4