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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Main Authors:	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
Format:	Article
Language:	English
Published:	BioMed Central 2018-03-01
Series:	BMC Genomics
Subjects:	SMRT sequencing Mitochondrial DNA Nuclear DNA Phylogenetics DNA barcoding PCR
Online Access:	http://link.springer.com/article/10.1186/s12864-018-4611-3

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spelling	doaj-art-2673b0441b68411da2726f9fee8906a02018-08-16T00:49:34ZengBioMed CentralBMC 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
institution	Open Data Bank
collection	Open Access Journals
building	Directory of Open Access Journals
language	English
format	Article
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	BioMed Central
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
_version_	1612696500236713984

A Sequel to Sanger: amplicon sequencing that scales

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