Critical factors for assembling a high volume of DNA barcodes

Abstract

Large-scale DNA barcoding projects are now moving toward activation while the creation of a comprehensive barcode library for eukaryotes will ultimately require the acquisition of some 100 million barcodes. To satisfy this need, analytical facilities must adopt protocols that can support the rapid, cost-effective assembly of barcodes. In this paper we discuss the prospects for establishing high volume DNA barcoding facilities by evaluating key steps in the analytical chain from specimens to barcodes. Alliances with members of the taxonomic community represent the most effective strategy for provisioning the analytical chain with specimens. The optimal protocols for DNA extraction and subsequent PCR amplification of the barcode region depend strongly on their condition, but production targets of 100K barcode records per year are now feasible for facilities working with compliant specimens. The analysis of museum collections is currently challenging, but PCR cocktails that combine polymerases with repair enzyme(s) promise future success. Barcode analysis is already a cost-effective option for species identification in some situations and this will increasingly be the case as reference libraries are assembled and analytical protocols are simplified.

Figure 1

Typical specimen types and sizes used for DNA barcoding analysis as compared to a pencil head. (a), a lepidopteran leg; (b), a Daphnia; (c), a feather; (d), muscle tissue.

Figure 2

Evaluation of DNA isolation methods for high volume DNA barcoding analysis in different types of specimens. Five DNA isolation methods were compared for the amplification of full-length (∼650 bp) cox1 barcode sequence, by examining (a), % PCR success and (b), % sequencing success. Numbers on columns indicate cases of contamination.

Figure 3

Evaluation of different PCR enzymes for the amplification of cox1 for recent and archival specimens. Four enzymes including Taq polymerase (Taq), Restorase (Res), AccuTaq (Acc) and Diamond polymerase (Dia) were compared for the amplification of full-length and partial cox1 barcodes (658, 407, 155 bp). Results are shown as % PCR success.

Figure 4

Effectiveness of four PCR enzymes for the amplification of cox1 in archived specimens of varied ages. Taq polymerase (Taq), Restorase (Res), AccuTaq (Acc) and Diamond polymerase (Dia) were compared for the amplification of full and partial cox1 barcodes (658, 407,155 bp) in archival moth specimens from six age groups (2, 4, 8, 16, ∼32, ∼64 years). Results are shown as (a), % PCR success and (b), % sequencing success.