A Sequel to Sanger: Amplicon Sequencing That Scales
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.
Ratnasingham Sujeevan、deWaard Jeremy R、Ivanova Natalia V、Zakharov Evgeny V、Janzen Daniel H、Prosser Sean WJ、Hebert Paul DN、Hallwachs Winnie、Braukmann Thomas WA、Sones Jayme E、Naik Suresh
Centre 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 PennsylvaniaCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphDepartment of Biology, University of PennsylvaniaCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of GuelphCentre for Biodiversity Genomics, University of Guelph
分子生物学生物科学研究方法、生物科学研究技术遗传学
SMRT sequencingmitochondrial DNAnuclear DNAphylogeneticsDNA barcodingPCRnucleotide compositionhomopolymer
Ratnasingham Sujeevan,deWaard Jeremy R,Ivanova Natalia V,Zakharov Evgeny V,Janzen Daniel H,Prosser Sean WJ,Hebert Paul DN,Hallwachs Winnie,Braukmann Thomas WA,Sones Jayme E,Naik Suresh.A Sequel to Sanger: Amplicon Sequencing That Scales[EB/OL].(2025-03-28)[2025-05-24].https://www.biorxiv.org/content/10.1101/191619.点此复制
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