sanger.pdf - Hebert et al BMC Genomics(2018 19:219...

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METHODOLOGY ARTICLE Open Access A Sequel to Sanger: amplicon sequencing that scales Paul D. N. Hebert 1* , Thomas W. A. Braukmann 1 , Sean W. J. Prosser 1 , Sujeevan Ratnasingham 1 , Jeremy R. deWaard 1 , Natalia V. Ivanova 1 , Daniel H. Janzen 2 , Winnie Hallwachs 2 , Suresh Naik 1 , Jayme E. Sones 1 and Evgeny V. Zakharov 1 Abstract Background: 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. Results: By examining templates from more than 5000 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 can reduce greatly reduce sequencing costs in comparison to first (Sanger) and second generation platforms (Illumina, Ion). Conclusions: SMRT analysis generates high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts. Analytical costs are low, substantially less than those for first or second generation sequencers. When implemented on the SEQUEL platform, SMRT analysis enables massive amplicon characterization because each instrument can recover sequences from more than 5 million DNA extracts a year. Keywords: SMRT sequencing, Mitochondrial DNA, Nuclear DNA, Phylogenetics, DNA barcoding, PCR, Nucleotide composition, Homopolymer Background High-throughput sequencers are doubling their analytical capacity every 9 months [ 1 , 2 ], but their reads are gener- ally short (< 400 bp) and error rates reach 0.8% 1.7% [ 3 ]. These limitations are an important constraint in three contexts: de novo genome assemblies are difficult [ 4 ], complex regions of well-known genomes can be intract- able [ 5 ], and sequencing long amplicons is inefficient. Be- cause of the latter constraint, Sanger sequencing is still widely used for amplicon characterization [ 6 9 ] despite its relatively high cost [ 10 ]. While recent studies have established that Illumina [ 11 , 12 ] and Ion Torrent [ 10 ] platforms can analyze 1 kb amplicons with good accuracy, their need to concatenate short reads creates risks to data quality linked to the recovery of chimeras and pseudo- genes. As well, because of their relatively complex work- flows, costs are only three to four times less than those for Sanger analysis.
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