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Data from: A sequel to Sanger: amplicon sequencing that scales
Hebert, Paul D.N.; Braukmann, Thomas W.A.; Prosser, Sean W.J.; Ratnasingham, Sujeevan; deWaard, Jeremy R.; Ivanova, Natalia V.; Janzen, Daniel H.; Hallwachs, Winnie; Naik, Suresh; Sones, Jayme E.; Zakharov, Evgeny V. — 2019-01-12 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. https://creativecommons.org/publicdomain/zero/1.0/
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Data from: Next-generation DNA barcoding: using next-generation sequencing to enhance and accelerate DNA barcode capture from single specimens
Shokralla, Shadi; Gibson, Joel F.; Nikbakht, Hamid; Janzen, Daniel H.; Hallwachs, Winnie; Hajibabaei, Mehrdad — 2014-01-27 DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large-scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next-generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high-target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next-generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10-mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full-length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full-length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next-generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.
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Data from: Massively parallel multiplex DNA sequencing for specimen identification using an Illumina MiSeq platform
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Shokralla, Shadi; Porter, Teresita M.; Gibson, Joel F.; Dobosz, Rafal; Janzen, Daniel H.; Hallwachs, Winnie; Golding, G. Brian; Hajibabaei, Mehrdad — 2016-02-18 Genetic information is a valuable component of biosystematics, especially specimen identification through the use of species-specific DNA barcodes. Although many genomics applications have shifted to High-Throughput Sequencing (HTS) or Next-Generation Sequencing (NGS) technologies, sample identification (e.g., via DNA barcoding) is still most often done with Sanger sequencing. Here, we present a scalable double dual-indexing approach using an Illumina Miseq platform to sequence DNA barcode markers. We achieved 97.3% success by using half of an Illumina Miseq flowcell to obtain 658 base pairs of the cytochrome c oxidase I DNA barcode in 1,010 specimens from eleven orders of arthropods. Our approach recovers a greater proportion of DNA barcode sequences from individuals than does conventional Sanger sequencing, while at the same time reducing both per specimen costs and labor time by nearly 80%. In addition, the use of HTS allows the recovery of multiple sequences per specimen, for deeper analysis of genetic variation in target gene regions.

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(auteur : Janzen, Daniel H.)

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