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

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

Søren M Karst #  1 , Ryan M Ziels #  2 , Rasmus H Kirkegaard  1 , Emil A Sørensen  1 , Daniel McDonald  3 , Qiyun Zhu  3 , Rob Knight  3   4   5   6 , Mads Albertsen  7
Affiliations

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

Søren M Karst et al. Nat Methods. 2021 Feb.

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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