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

Research output: Contribution to journal › Article › peer-review

146 Scopus citations

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 (US)
Pages (from-to)	165-169
Number of pages	5
Journal	Nature Methods
Volume	18
Issue number	2
DOIs	https://doi.org/10.1038/s41592-020-01041-y
State	Published - Feb 2021
Externally published	Yes

ASJC Scopus subject areas

Biotechnology
Biochemistry
Molecular Biology
Cell Biology

Access to Document

10.1038/s41592-020-01041-y

Cite this

@article{16d2845e7cc245968797547175f162be,

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.",

author = "Karst, {S{\o}ren M.} and Ziels, {Ryan M.} and Kirkegaard, {Rasmus H.} and S{\o}rensen, {Emil A.} and Daniel McDonald and Qiyun Zhu and Rob Knight and Mads Albertsen",

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: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.",

year = "2021",

month = feb,

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

language = "English (US)",

volume = "18",

pages = "165--169",

journal = "Nature Methods",

issn = "1548-7091",

publisher = "Nature Publishing Group",

number = "2",

}

TY - JOUR

T1 - High-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

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

N1 - 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.

PY - 2021/2

Y1 - 2021/2

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.

UR - http://www.scopus.com/inward/record.url?scp=85100101493&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85100101493&partnerID=8YFLogxK

U2 - 10.1038/s41592-020-01041-y

DO - 10.1038/s41592-020-01041-y

M3 - Article

C2 - 33432244

AN - SCOPUS:85100101493

SN - 1548-7091

VL - 18

SP - 165

EP - 169

JO - Nature Methods

JF - Nature Methods

IS - 2

ER -

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

Abstract

ASJC Scopus subject areas

Access to Document

Other files and links

Fingerprint

Cite this