Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

Abstract

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.

DNA barcoding mosquitoes: advice for potential prospectors

“…These methods have been assessed and compared with traditional Sanger sequencing and found to be both superior and more efficient in terms of labour and cost (Shokralla et al 2014;Shokralla et al 2015;Batovska et al 2017).…”

Towards global interoperability for supporting biodiversity research on essential biodiversity variables (EBVs)

“…For instance, the species concept is very elusive for the microbial world, where the strain/isolate definition might be more practically helpful to allow exhaustive description of phenotypic features (Ahn, Chai, and Pan 2014;Caro-Quintero and Konstantinidis 2012). This can be potentially addressed through specific and progressively advanced molecular methods such as DNA barcoding and metagenomics (Gilbert and Dupont 2011;Kress et al 2015;Shokralla et al 2014;Simon and Daniel 2011).…”

“…It also includes molecular biodiversity data (e.g. barcoding, meta-barcoding and metagenomic approaches based on next-generation sequencing technologies and bioinformatic data analysis) which increasingly allow species or strain identification at a high rate and accuracy, even in the most remote and extreme ecosystems (Gilbert and Dupont 2011;Kress et al 2015;Shokralla et al 2014;Simon and Daniel 2011). Workshop 3 will be centred on the EBV class 'Species traits' which can include phenology, plant functional traits, animal body mass, natal dispersal distances or migratory behaviour (Pereira et al 2013).…”

Misidentification of bluefin tuna larvae: a call for caution and taxonomic reform

“…Researchers are now beginning to use aspects of each approach that optimize their resource costs: use of voucher specimens identified by a morphology-based approach, NGS techniques to discover new barcoding sites or diagnostic/informative loci, followed by Sanger sequencing of low numbers of samples or genotyping of high numbers of individuals with microarrays (Shokralla et al 2014). …”