Sequencing 16S rRNA gene fragments using the PacBio SMRT DNA sequencing system

Abstract

Over the past 10 years, microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted highly parallelized sequencing platforms. These new platforms, such as 454 and Illumina’s MiSeq, have allowed researchers to obtain millions of high quality but short sequences. The result of the added sequencing depth has been significant improvements in experimental design. The tradeoff has been the decline in the number of full-length reference sequences that are deposited into databases. To overcome this problem, we tested the ability of the PacBio Single Molecule, Real-Time (SMRT) DNA sequencing platform to generate sequence reads from the 16S rRNA gene. We generated sequencing data from the V4, V3–V5, V1–V3, V1–V5, V1–V6, and V1–V9 variable regions from within the 16S rRNA gene using DNA from a synthetic mock community and natural samples collected from human feces, mouse feces, and soil. The mock community allowed us to assess the actual sequencing error rate and how that error rate changed when different curation methods were applied. We developed a simple method based on sequence characteristics and quality scores to reduce the observed error rate for the V1–V9 region from 0.69 to 0.027%. This error rate is comparable to what has been observed for the shorter reads generated by 454 and Illumina’s MiSeq sequencing platforms. Although the per base sequencing cost is still significantly more than that of MiSeq, the prospect of supplementing reference databases with full-length sequences from organisms below the limit of detection from the Sanger approach is exciting.

Preprinting Microbiology

“…Instead of ignoring this feedback and resubmitting the paper to address the reviews, we generated new data and submitted an updated preprint a year later with a simultaneous submission to PeerJ that incorporated the original reviews and the feedback we received from the academic scientist and Pacific Biosciences. It was eventually published by PeerJ (42, 43). Since 2015, we have continued to post papers as preprints at the same time as we have submitted papers.…”

Review of Molecular Techniques for the Identification of Bacterial Communities in Biological Effluent Treatment Facilities at Pulp and Paper Mills

“…Microbial ecologists have largely abandoned sequencing 16S rRNA genes by the Sanger sequencing method and have instead adopted new parallelized sequencing (Schloss et al 2016). The advent of DNA sequencing techniques in past years has far exceeded expectations (Ansorge et al 2017).…”

Long‐read metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

“…PacBio has lower throughput and higher error rates than Illumina but can produce reads that are over 20 kb long at a fraction of the cost of Sanger sequencing. In the last few years, PacBio sequencing has started to be applied to metabarcoding studies, primarily on prokaryotic 16S rDNA (Mosher et al, 2014;Schloss, Jenior, Koumpouras, Westcott, & Highlander, 2016;Wagner et al, 2016) and most recently on larger amplicons also including the 23S rDNA (Martijn et al, 2019). For eukaryotes, the 18S rDNA was nearly fully sequenced for targeted microbial groups (Orr et al, 2018), whilst longer regions also spanning the ITS and the large subunit (LSU) 28S gene were used to analyse fungal diversity (Heeger et al, 2018;Tedersoo & Anslan, 2019;Tedersoo, Tooming-Klunderud, & Anslan, 2018).…”