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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.
Bibliographical notePublisher Copyright:
© 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.
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Albertsen, M., Nielsen, P. H., Jensen, T. B. N., Sørensen, E. A., Sereika, M., Dottorini, G., Petriglieri, F., Jørgensen, V. R., Kirkegaard, R. H., Yang, Y., Karst, S. M., Giguere, A. T., Knutsson, S., Singleton, C. M., Dueholm, M. K. D. & Mølvang Dall, S.
01/01/2019 → 31/12/2025
01/01/2017 → 01/01/2022