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 journalJournal articleResearchpeer-review

78 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 languageEnglish
JournalNature Methods
Volume18
Issue number2
Pages (from-to)165-169
Number of pages5
ISSN1548-7091
DOIs
Publication statusPublished - Feb 2021

Bibliographical note

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

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