A Sequel to Sanger: amplicon sequencing that scales
Paul D. N. Hebert,Thomas Braukmann,Sean W. J. Prosser,Sujeevan Ratnasingham,Jeremy R deWaard,Natalia Ivanova,Daniel H. Janzen,Winnie Hallwachs,Suresh Naik,Jayme E Sones,Evgeny Zakharov +10 more
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TLDR
The capacity of single molecule, real-time (SMRT) sequencing implemented on the SEQUEL platform to overcome limitations and generate high-fidelity sequences from amplicons with varying GC content and is resilient to homopolymer tracts is tested.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 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). 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.read more
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Accurate circular consensus long-read sequencing improves variant detection and assembly of a human genome.
Aaron M. Wenger,Paul Peluso,William J Rowell,Pi-Chuan Chang,Richard Hall,Gregory T. Concepcion,Jana Ebler,Arkarachai Fungtammasan,Alexander Kolesnikov,Nathan D. Olson,Armin Töpfer,Michael Alonge,Medhat Mahmoud,Yufeng Qian,Chen-Shan Chin,Adam M. Phillippy,Michael C. Schatz,Gene Myers,Mark A. DePristo,Jue Ruan,Tobias Marschall,Tobias Marschall,Fritz J. Sedlazeck,Justin M. Zook,Heng Li,Sergey Koren,Andrew Carroll,David R. Rank,Michael W. Hunkapiller +28 more
TL;DR: The optimization of circular consensus sequencing (CCS) is reported to improve the accuracy of single-molecule real-time (SMRT) sequencing (PacBio) and generate highly accurate (99.8%) long high-fidelity (HiFi) reads with an average length of 13.5 kilobases (kb).
Journal ArticleDOI
Long-Read Sequencing Emerging in Medical Genetics
TL;DR: The current LRS-based research on human genetic disorders is summarized and the potential of these technologies to facilitate the next major advancements in medical genetics is discussed.
Journal ArticleDOI
High-throughput amplicon sequencing of the full-length 16S rRNA gene with single-nucleotide resolution.
Benjamin J. Callahan,Joan Wong,Cheryl Heiner,Steve Oh,Casey M. Theriot,Ajay S. Gulati,Sarah K. McGill,Michael K. Dougherty +7 more
TL;DR: A high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate is presented.
Journal ArticleDOI
Real-time DNA barcoding in a rainforest using nanopore sequencing: opportunities for rapid biodiversity assessments and local capacity building.
Aaron Pomerantz,Nicolás Peñafiel,Alejandro Arteaga,Lucas Bustamante,Frank Pichardo,Luis A. Coloma,César L. Barrio-Amorós,David Salazar-Valenzuela,Stefan Prost,Stefan Prost +9 more
TL;DR: It is demonstrated that nanopore sequencing can be implemented in a remote tropical forest to quickly and accurately identify species using DNA barcoding, as it generated consensus sequences for species resolution with an accuracy of >99% in less than 24 hours after collecting specimens.
Journal ArticleDOI
Somatic APP gene recombination in Alzheimer’s disease and normal neurons
Ming-Hsiang Lee,Benjamin Siddoway,Gwendolyn E. Kaeser,Gwendolyn E. Kaeser,Igor Segota,Richard Rivera,William J. Romanow,Christine S. Liu,Christine S. Liu,Chris Park,Chris Park,Grace Kennedy,Tao Long,Jerold Chun +13 more
TL;DR: For example, this article described recombination of the Alzheimer's disease-related gene APP in human neurons, occurring mosaically as thousands of variant "genomic cDNAs" (gencDNAs).
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