Karst Søren M, Ziels Ryan M, Kirkegaard Rasmus H, Sørensen Emil A, McDonald Daniel, Zhu Qiyun, Knight Rob, Albertsen Mads
Center for Microbial Communities, Department of Chemistry and Bioscience, Aalborg University, Aalborg, Denmark.
Department of Civil Engineering, The University of British Columbia, Vancouver, British Columbia, Canada.
Nat Methods. 2021 Feb;18(2):165-169. doi: 10.1038/s41592-020-01041-y. Epub 2021 Jan 11.
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.
对于短读长技术而言，对大基因组区域进行高通量扩增子测序仍然具有挑战性。在此，我们报告了一种高通量扩增子测序方法，该方法将独特分子标识符（UMIs）与牛津纳米孔技术（ONT）或太平洋生物科学公司的环形一致序列测序相结合，从而产生大基因组区域的高精度单分子一致序列。我们将我们的方法应用于对参考微生物群落的核糖体RNA操纵子扩增子（约4500 bp）和基因组序列（>10000 bp）进行测序，在这些群落中我们观察到嵌合体率<0.02%。为了使UMI一致错误率<0.01%，需要15×（ONT R10.3）、25×（ONT R9.4.1）和3×（太平洋生物科学公司的环形一致序列测序）的UMI读段覆盖度，它们分别提供了0.0042%、0.0041%和0.0007%的平均错误率。
