Enabling high-accuracy long-read amplicon sequences using unique molecular identifers and Nanopore sequencing

High-throughput amplicon sequencing of large genomic regions represents a challenge for existing short-read technologies. Long-read technologies can, in theory, sequence large genomic regions, but they currently suffer from high error rates. Here, we report a high-throughput amplicon sequencing approach that combines unique molecular identifiers (UMIs) with Oxford Nanopore sequencing to generate single-molecule consensus sequences of large genomic regions. We demonstrate the approach by generating nearly 10,000 full-length ribosomal RNA (rRNA) operons of roughly 4,400 bp in length from a mock microbial community consisting of 8 bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.05%, with no detectable chimeras due to a rigorous UMI-barcode filtering strategy. The simplicity and accessibility of this method paves way for widespread use of high-accuracy amplicon sequencing in a variety of genomic applications.针对大片段基因组区域的高通量扩增子测序，是现有短读长测序技术面临的一大挑战。长读长测序技术理论上可对大片段基因组区域进行测序，但目前仍存在较高的错误率。本研究报道了一种高通量扩增子测序方法，该方法将唯一分子标识符（Unique Molecular Identifiers, UMIs）与牛津纳米孔（Oxford Nanopore）测序技术相结合，可生成大片段基因组区域的单分子一致性序列。我们通过单张牛津纳米孔MinION测序流动槽，从由8种细菌组成的模拟微生物群落中，获得了近10000条长度约4400 bp的完整核糖体RNA（ribosomal RNA, rRNA）操纵子序列，以此验证了该方法的有效性。该方法生成的一致性序列平均错误率仅为0.05%，且由于采用了严格的UMI-条形码过滤策略，未检测到嵌合序列。该方法操作简便、易于获取，为高精度扩增子测序在各类基因组学应用中的广泛应用铺平了道路。