ena-STUDY-AALBORG UNIVERSITY-10-08-2020-12:59:51:991-2233
Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing
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 CCS (PacBio) sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence rRNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities. We observed a chimera rate below 0.02%, and to reach a mean UMI consensus error rate of <0.01% (Q40) a UMI read coverage of 15x (ONT R10.3), 25x (ONT 9.4.1) and 3x (PacBio CCS) was needed, above which the corresponding mean error rates were 0.0042%, 0.0041%, and 0.0007%, respectively.
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 CCS (PacBio) sequencing, yielding high accuracy single-molecule consensus sequences of large genomic regions. We applied our approach to sequence rRNA operon amplicons (~4,500 bp) and genomic sequences (>10,000 bp) of reference microbial communities. We observed a chimera rate below 0.02%, and to reach a mean UMI consensus error rate of <0.01% (Q40) a UMI read coverage of 15x (ONT R10.3), 25x (ONT 9.4.1) and 3x (PacBio CCS) was needed, above which the corresponding mean error rates were 0.0042%, 0.0041%, and 0.0007%, respectively.