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

Nat Methods. 2021 Feb;18(2):165-169. doi: 10.1038/s41592-020-01041-y. Epub 2021 Jan 11.

Authors

Søren M Karst^#¹, Ryan M Ziels^#², Rasmus H Kirkegaard¹, Emil A Sørensen¹, Daniel McDonald³, Qiyun Zhu³, Rob Knight^{3

4

5

6}, Mads Albertsen⁷

Affiliations

¹ Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.
² Department of Civil Engineering, The University of British Columbia, Vancouver, British Columbia, Canada.
³ Department of Pediatrics, University of California San Diego, La Jolla, CA, USA.
⁴ Department of Bioengineering, University of California San Diego, La Jolla, CA, USA.
⁵ Center for Microbiome Innovation, University of California San Diego, La Jolla, CA, USA.
⁶ Department of Computer Science and Engineering, University of California San Diego, La Jolla, CA, USA.
⁷ Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark. ma@bio.aau.dk.

^# Contributed equally.

PMID: 33432244
DOI: 10.1038/s41592-020-01041-y

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

High-Throughput Nucleotide Sequencing / methods*
Microbiota*
Nanopores*
Workflow