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

By Søren M. Karst, Ryan M. Ziels, Rasmus H. Kirkegaard, Mads Albertsen

Posted 24 May 2019
bioRxiv DOI: 10.1101/645903

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 eight bacterial species using a single Oxford Nanopore MinION flowcell. The mean error rate of the consensus sequences was 0.03%, 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.

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