Enabling high-accuracy long-read amplicon sequences using unique molecular identifiers with Nanopore or PacBio sequencing

Abstract

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 highthroughput 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.

Long-read sequencing to interrogate strain-level variation among adherent-invasiveEscherichia coliisolated from human intestinal tissue

“…Emerging singlemolecule (third-generation/long-read) sequencing technologies including Pacific Biosciences ("PacBio") and Oxford Nanopore Technologies ("nanopore") produce reads 10s to 100s of kilobases in length, often without prior amplification. Recent work [14][15][16][17] has demonstrated the utility of these longer reads for sequencing the entire 16S gene or entire rRNA operon, with corresponding increase in taxonomic resolution by capturing more variable sequence, including all nine variable regions of 16S, the internal transcribed spacers (ITS), and 23S. To support the extension of these approaches to characterize strain-level variation of tissue-associated (mucosaassociated) microbiota contributing to IBD and in models of experimental colitis in mice, with a particular focus on mechanistic studies of AIEC, we produced complete genome assemblies for eight AIEC and non-AIEC E. coli, describe the genomic variation among these strains, particularly within the rRNA operon, and demonstrate the accurate identification of these strains in mixed in vitro and in vivo microbiota.…”

Parallel genetics of regulatory sequencesin vivo

“…Our targeted sequencing protocol can capture long deletions, uses the same amplicon for the whole locus, and allows sample multiplexing. Unique molecule counting methods 48 for long reads 49,50 could be incorporated to reduce PCR biases. Established protocols 28 are available for shorter (100-300 bp) target regions.…”

A sample-to-report solution for taxonomic identification of cultured bacteria in the clinical setting based on nanopore sequencing

“…Even if databases contained sequences with up to 99% identity to the analysed species, further improvements could often be made by adding closer reference sequences ( Figure 6). When the consensus sequence was constructed, however, taxonomic identification based on the obtained consensus sequence was far less sensitive to database relied on rather laborious wet-lab procedures such as rolling cycle amplification or unique tagging of the individual amplicons before sequencing [25,26]. Unlike previous studies we specifically designed our workflow for clinical routine applications.…”

“…One obstacle for a broad adoption of nanopore sequencing in routine diagnostic laboratories is the added bioinformatic complexity as compared to established Sanger sequencing workflows. Furthermore, available workflows are often limited to the analysis of pure amplicons [20][21][22][23], include complex modifications of the ONT laboratory workflows [25,26], or lack published validation by using samples other than mock communities [27,28].…”