DNA barcodes from century‐old type specimens using next‐generation sequencing

Abstract

Type specimens have high scientific importance because they provide the only certain connection between the application of a Linnean name and a physical specimen. Many other individuals may have been identified as a particular species, but their linkage to the taxon concept is inferential. Because type specimens are often more than a century old and have experienced conditions unfavourable for DNA preservation, success in sequence recovery has been uncertain. This study addresses this challenge by employing next-generation sequencing (NGS) to recover sequences for the barcode region of the cytochrome c oxidase 1 gene from small amounts of template DNA. DNA quality was first screened in more than 1800 century-old type specimens of Lepidoptera by attempting to recover 164-bp and 94-bp reads via Sanger sequencing. This analysis permitted the assignment of each specimen to one of three DNA quality categories -high (164-bp sequence), medium (94-bp sequence) or low (no sequence). Ten specimens from each category were subsequently analysed via a PCR-based NGS protocol requiring very little template DNA. It recovered sequence information from all specimens with average read lengths ranging from 458 bp to 610 bp for the three DNA categories. By sequencing ten specimens in each NGS run, costs were similar to Sanger analysis. Future increases in the number of specimens processed in each run promise substantial reductions in cost, making it possible to anticipate a future where barcode sequences are available from most type specimens.

Calibrating the taxonomy of a megadiverse insect family: 3000 DNA barcodes from geometrid type specimens (Lepidoptera, Geometridae)

“…There was 100% concordance between sequences generated from the same specimen using NGS and Sanger sequencing for the 20 type specimens examined by Prosser et al (2016). Our extended data set (93 type specimens) confirms this result.…”

“…For the geometrid types from NHM London, the quality control of the sequences which were generated in parallel from the same DNA extracts with the Sanger approach and with NGS revealed a 100% match in all cases (Prosser et al 2016). The same study showed that all DNA sequences from century-old type specimens perfectly matched (100%) sequences from recently collected specimens when these were available in the Barcode of Life Data Systems, BOLD (Ratnasingham and Hebert 2007).…”

“…2, 3). By comparison, sequence recovery via NGS (Prosser et al 2016) was much higher for the 101 old specimens with 100% in recovering a sequence with read lengths ranging from 213 to 610 bp (after excluding nucleotide positions labeled as "N"), even when Sanger sequencing failed completely. In total, 94% of the sequences were longer than 400 bp.…”

“…(3) Full-length barcodes were recovered from more than 200 century-old specimens using Sanger sequence analysis of multiple short amplicons (Tables 3, 4; for details of the protocol see Lees et al 2010). A NGSbased approach (Prosser et al 2016) was also used to recover full-length barcodes from 101 century-old Geometridae specimens from the NHM (92) and SNSB-ZSM (9), see Fig. 1.…”

“…These analyses initially focused on the recovery of short fragments, 164 base pair (bp) and (or) 94 bp from the middle of the COI barcode fragment, as these allow unambiguous species identification in more than 90% of cases (Meusnier et al 2008) and reliable connection to fulllength (658 bp) DNA barcodes from other individuals of the same species ). However, work was also directed toward the development of a protocol employing multiplex PCR to generate short amplicons for analysis using Next-Generation-Sequencing (NGS) to recover the full 658-bp barcode region (Prosser et al 2016;see Speidel et al 2015 for a recent study on North African lasiocampids involving a 194-year-old type specimen). This new approach is particularly valuable in gaining sequence information for the many species which are only known from type material and for resolving cases involving very closely related species.…”