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^*

^*Corresponding author for this work

Research output: Contribution to journal › Journal article › Research › peer-review

192 Citations (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.

Original language	English
Journal	Nature Methods
Volume	18
Issue number	2
Pages (from-to)	165-169
Number of pages	5
ISSN	1548-7091
DOIs	https://doi.org/10.1038/s41592-020-01041-y
Publication status	Published - Feb 2021

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© 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.pdfLicence: CC BY 4.0

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

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.",

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doi = "10.1038/s41592-020-01041-y",

language = "English",

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AU - Karst, Søren M.

AU - Ziels, Ryan M.

AU - Kirkegaard, Rasmus H.

AU - Sørensen, Emil A.

AU - McDonald, Daniel

AU - Zhu, Qiyun

AU - Knight, Rob

AU - Albertsen, Mads

PY - 2021/2

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N2 - 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.

AB - 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.

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JO - Nature Methods

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

Abstract

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

Abstract

Bibliographical 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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