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.

大基因组区域的高通量扩增子测序对于短读技术仍然具有挑战性。在这里，我们报告了一种高通量扩增子测序方法，该方法将独特的分子标识符 (UMI) 与牛津纳米孔技术 (ONT) 或 Pacific Biosciences 循环共有测序相结合，产生了大基因组区域的高精度单分子共有序列。我们应用我们的方法对参考微生物群落的核糖体 RNA 操纵子扩增子 (~4,500 bp) 和基因组序列 (>10,000 bp) 进行测序，其中我们观察到嵌合率 <0.02%。为了达到平均UMI共识错误率<0.01%，需要15×（ONT R10.3）、25×（ONT R9.4.1）和3×（太平洋生物科学循环共识测序）的UMI读取覆盖率，这提供了一个平均错误率分别为 0.0042%、0.0041% 和 0.0007%。