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 journalArticlepeer-review

7 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 languageEnglish (US)
Pages (from-to)165-169
Number of pages5
JournalNature Methods
Volume18
Issue number2
DOIs
StatePublished - Feb 2021
Externally publishedYes

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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