PT  - JOURNAL ARTICLE
AU  - Paul DN Hebert
AU  - Thomas WA Braukmann
AU  - Sean WJ Prosser
AU  - Sujeevan Ratnasingham
AU  - Jeremy R deWaard
AU  - Natalia V Ivanova
AU  - Daniel H Janzen
AU  - Winnie Hallwachs
AU  - Suresh Naik
AU  - Jayme E Sones
AU  - Evgeny V Zakharov
TI  - A Sequel to Sanger: Amplicon Sequencing That Scales
AID  - 10.1101/191619
DP  - 2017 Jan 01
TA  - bioRxiv
PG  - 191619
4099  - http://biorxiv.org/content/early/2017/09/20/191619.short
4100  - http://biorxiv.org/content/early/2017/09/20/191619.full
AB  - 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.