A Sequel to Sanger: amplicon sequencing that scales
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) seq...
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doaj-2673b0441b68411da2726f9fee8906a02020-11-25T01:31:21ZengBMCBMC Genomics1471-21642018-03-0119111410.1186/s12864-018-4611-3A Sequel to Sanger: amplicon sequencing that scalesPaul D. N. Hebert0Thomas W. A. Braukmann1Sean W. J. Prosser2Sujeevan Ratnasingham3Jeremy R. deWaard4Natalia V. Ivanova5Daniel H. Janzen6Winnie Hallwachs7Suresh Naik8Jayme E. Sones9Evgeny V. Zakharov10Centre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphDepartment of Biology, University of PennsylvaniaDepartment of Biology, University of PennsylvaniaCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphAbstract 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.http://link.springer.com/article/10.1186/s12864-018-4611-3SMRT sequencingMitochondrial DNANuclear DNAPhylogeneticsDNA barcodingPCR |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Paul D. N. Hebert Thomas W. A. Braukmann Sean W. J. Prosser Sujeevan Ratnasingham Jeremy R. deWaard Natalia V. Ivanova Daniel H. Janzen Winnie Hallwachs Suresh Naik Jayme E. Sones Evgeny V. Zakharov |
spellingShingle |
Paul D. N. Hebert Thomas W. A. Braukmann Sean W. J. Prosser Sujeevan Ratnasingham Jeremy R. deWaard Natalia V. Ivanova Daniel H. Janzen Winnie Hallwachs Suresh Naik Jayme E. Sones Evgeny V. Zakharov A Sequel to Sanger: amplicon sequencing that scales BMC Genomics SMRT sequencing Mitochondrial DNA Nuclear DNA Phylogenetics DNA barcoding PCR |
author_facet |
Paul D. N. Hebert Thomas W. A. Braukmann Sean W. J. Prosser Sujeevan Ratnasingham Jeremy R. deWaard Natalia V. Ivanova Daniel H. Janzen Winnie Hallwachs Suresh Naik Jayme E. Sones Evgeny V. Zakharov |
author_sort |
Paul D. N. Hebert |
title |
A Sequel to Sanger: amplicon sequencing that scales |
title_short |
A Sequel to Sanger: amplicon sequencing that scales |
title_full |
A Sequel to Sanger: amplicon sequencing that scales |
title_fullStr |
A Sequel to Sanger: amplicon sequencing that scales |
title_full_unstemmed |
A Sequel to Sanger: amplicon sequencing that scales |
title_sort |
sequel to sanger: amplicon sequencing that scales |
publisher |
BMC |
series |
BMC Genomics |
issn |
1471-2164 |
publishDate |
2018-03-01 |
description |
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. |
topic |
SMRT sequencing Mitochondrial DNA Nuclear DNA Phylogenetics DNA barcoding PCR |
url |
http://link.springer.com/article/10.1186/s12864-018-4611-3 |
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