Abstract
Over the past decade, biodiversity researchers have dedicated tremendous efforts to constructing DNA reference barcodes for rapid species registration and identification. Although analytical cost for standard DNA barcoding has been significantly reduced since early 2000, further dramatic reduction in barcoding costs is unlikely because Sanger sequencing is approaching its limits in throughput and chemistry cost. Constraints in barcoding cost not only led to unbalanced barcoding efforts around the globe, but also prevented high-throughput sequencing (HTS)–based taxonomic identification from applying binomial species names, which provide crucial linkages to biological knowledge. We developed an Illumina-based pipeline, HIFI-Barcode, to produce full-length Cytochrome c oxidase subunit I (COI) barcodes from pooled polymerase chain reaction amplicons generated by individual specimens. The new pipeline generated accurate barcode sequences that were comparable to Sanger standards, even for different haplotypes of the same species that were only a few nucleotides different from each other. Additionally, the new pipeline was much more sensitive in recovering amplicons at low quantity. The HIFI-Barcode pipeline successfully recovered barcodes from more than 78% of the polymerase chain reactions that didn’t show clear bands on the electrophoresis gel. Moreover, sequencing results based on the single molecular sequencing platform Pacbio confirmed the accuracy of the HIFI-Barcode results. Altogether, the new pipeline can provide an improved solution to produce full-length reference barcodes at about one-tenth of the current cost, enabling construction of comprehensive barcode libraries for local fauna, leading to a feasible direction for DNA barcoding global biomes.
Highlights
IntroductionBiodiversity research has seen paradigm shifts in methodology developments and applications [1], where standard DNA sequences, e.g., DNA barcodes, are adopted for fast and accurate taxonomic diagnoses and high-throughput sequencing (HTS) platforms are employed in analysis of complex biological samples, including bulk samples [2, 3], environmental DNA (eDNA [4]), invertebrate-derived DNA (iDNA [5, 6]), etc
Biodiversity research has seen paradigm shifts in methodology developments and applications [1], where standard DNA sequences, e.g., DNA barcodes, are adopted for fast and accurate taxonomic diagnoses and high-throughput sequencing (HTS) platforms are employed in analysis of complex biological samples, including bulk samples [2, 3], environmental DNA, invertebrate-derived DNA, etc
Two 96-well plates were prepared for the current work: 1 plate containing 96 high-quality lepidopteran DNA was used to evaluate the accuracy of our HTS method using Sanger barcodes as the gold standard; a second plate containing 95 randomly selected DNA regardless of quality and polymerase chain reaction (PCR) yields plus a blank control was prepared to examine the success rate of our HTS method compared with the classic Sanger approach
Summary
Biodiversity research has seen paradigm shifts in methodology developments and applications [1], where standard DNA sequences, e.g., DNA barcodes, are adopted for fast and accurate taxonomic diagnoses and high-throughput sequencing (HTS) platforms are employed in analysis of complex biological samples, including bulk samples [2, 3], environmental DNA (eDNA [4]), invertebrate-derived DNA (iDNA [5, 6]), etc. Boosted by HTS technologies, DNA metabarcoding and mitochondrial metagenomics (mitochondrial genome skimming) have been applied in investigations of local biodiversity and in evaluation of biological managements [13,14,15,16,17]. A typical dilemma, is the lack of local barcode references from which HTS biodiversity analysis could draw conclusions on species occurrences This is primarily due to unbalanced barcoding efforts around the globe, where regions in desperate need for biodiversity research are typically suffering from insufficient funding for taxonomy work, especially for DNA-based studies. HTS-based taxonomic registrations are often constrained to applying molecular operational units (OTUs) instead of binomial species names, and are unable to associate existing biological and ecological knowledge to the resultant diversity composition
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