High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Søren M. Karst; Ryan M. Ziels; Rasmus H. Kirkegaard; Emil A. Sørensen; Daniel McDonald; Qiyun Zhu; Rob Knight; Mads Albertsen

doi:10.1038/s41592-020-01041-y

High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Søren M. Karst, Ryan M. Ziels, Rasmus H. Kirkegaard, Emil A. Sørensen, Daniel McDonald, Qiyun Zhu, Rob Knight, Mads Albertsen^*

^*Kontaktforfatter

Publikation: Bidrag til tidsskrift › Tidsskriftartikel › Forskning › peer review

146 Citationer (Scopus)

Abstract

High-throughput amplicon sequencing of large genomic regions remains challenging for short-read technologies. Here, we report a high-throughput amplicon sequencing approach combining unique molecular identifiers (UMIs) with Oxford Nanopore Technologies (ONT) or Pacific Biosciences circular consensus sequencing, yielding high-accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence ribosomal RNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities in which we observed a chimera rate <0.02%. To reach a mean UMI consensus error rate <0.01%, a UMI read coverage of 15× (ONT R10.3), 25× (ONT R9.4.1) and 3× (Pacific Biosciences circular consensus sequencing) is needed, which provides a mean error rate of 0.0042%, 0.0041% and 0.0007%, respectively.

Originalsprog	Engelsk
Tidsskrift	Nature Methods
Vol/bind	18
Udgave nummer	2
Sider (fra-til)	165-169
Antal sider	5
ISSN	1548-7091
DOI	https://doi.org/10.1038/s41592-020-01041-y
Status	Udgivet - feb. 2021

Bibliografisk note

Funding Information:
The study was funded by research grants from VILLUM FONDEN (15510) and the Poul Due Jensen Foundation (Microflora Danica). R.M.Z. was funded by grants from the Natural Sciences and Engineering Research Council of Canada (Discovery Grant) and Genome British Columbia (SIP011).

Publisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.

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10.1038/s41592-020-01041-y

https://www.biorxiv.org/content/biorxiv/early/2020/01/11/645903.full.pdfLicens: CC BY 4.0

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1 Afsluttet
1 Igangværende

Microflora Danica: The Microbiome of Denmark
Albertsen, M., Nielsen, P. H., Jensen, T. B. N., Sørensen, E. A., Sereika, M., Dottorini, G., Petriglieri, F., Jørgensen, V. R., Kirkegaard, R. H., Yang, Y., Karst, S. M., Giguere, A. T., Knutsson, S., Singleton, C. M., Dueholm, M. K. D., Mølvang Dall, S., Kristensen, J. M. & Delogu, F.
Poul Due Jensens Fond
01/01/2019 → 31/12/2025
Projekter: Projekt › Forskning
Deciphering the role of microbial dark matter by novel DNA sequencing approaches
Albertsen, M., Karst, S. M., Kirkegaard, R. H. & Yssing Michaelsen, T.
Villum Fonden
01/01/2017 → 01/01/2022
Projekter: Projekt › Forskning

Citationsformater

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High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Abstract

Bibliografisk note

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Microflora Danica: The Microbiome of Denmark

Deciphering the role of microbial dark matter by novel DNA sequencing approaches

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