Nanotechnology

16S ribosomal RNA

Amplicon

Biology

Gene

Nanopore

Nanopore sequencing

Identifier

Materials science

Programming language

Minion

Computer science

DNA sequencing

Computational biology

Amplicon sequencing

Polymerase chain reaction

Genetics

Nature Methods

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 (&gt;10,000 bp) of reference microbial communities in which we observed a chimera rate &lt;0.02%. To reach a mean UMI consensus error rate &lt;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. This work presents a sequencing strategy based on unique molecular identifiers that improves long-read consensus sequence accuracy of targeted amplicons as well as shotgun whole-genome fragments.

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