A Sequel to Sanger: Amplicon Sequencing That Scales

Abstract

Although high-throughput sequencers (HTS) have largely displaced their Sanger counterparts, the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing. The present study tests the capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome these limitations, employing 658 bp amplicons of the mitochondrial cytochrome c oxidase I gene as a model system. By examining templates from more than 5,000 species and 20,000 specimens, the performance of SMRT sequencing was tested with amplicons showing wide variation in GC composition and varied sequence attributes. SMRT and Sanger sequences were very similar, but SMRT sequencing provided more complete coverage, especially for amplicons with homopolymer tracts. Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis. Reflecting the capacity of each instrument to recover sequences from more than five million DNA extracts a year, this platform facilitates massive amplicon characterization.

High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution

“…However, amplicon sequencing applications are often sensitive to the presence of spurious sequence variants introduced by PCR and sequencing errors, and long-read sequencing has a much higher error rate (~10%) than does short-read sequencing (~0.5%). For PacBio, high long-read error rates can be ameliorated by the construction of circular consensus sequences (CCS), in which an amplified genetic locus is circularized and read through multiple times before a consensus sequence is reported (Hebert 2018). The CCS approach effectively trades read length for accuracy: raw reads reaching over 100 kilobases in length are processed to yield CCS reads that extend only 1-20 kilobases but that have a per-base accuracy comparable to that of short-read sequencing (Jiao 2013;Larsen 2014;Wenger 2019).…”

A Refined View of Airway Microbiome in Chronic Obstructive Pulmonary Disease at Species and Strain-levels

“…By generating long reads that extend tens of thousands of nucleotides, they offer the promise of increased taxonomic resolution by sequencing the full-length of 16S rRNA gene[13]. In these applications, the 16S amplicon is circularized and read through multiple passes before circular consensus sequences (CCS) is reported, which greatly reduced the initial high error rate (~10%) of the long-read sequencing to that comparable to short-read sequencing (~0.5%)[14, 15]. Recent development of sophisticated denoising algorithms further enable accurate bacterial species identification at single-nucleotide resolution with near-zero error rate[16].…”

Multiple-site diversification of regulatory sequences enables inter-species operability of genetic devices

“…To this end, the entire DNA constructs were subject to Pacific Biosciences SMRT (Single Molecule Real-Time) PCR amplicon sequencing, based on a modified version of Nyerges et al . 12,23,24 , as explained in Materials and Methods. The 5 analyzed samples included libraries Rv-A (sample 1) and Rv-B (sample 2), sorted libraries Rv-A (sample 3) and Rv-B (sample 4) and the 86 best performing clones (sample 5; Fig.…”