Pyrosequencing for Mini-Barcoding of Fresh and Old Museum Specimens

Abstract

DNA barcoding is an effective approach for species identification and for discovery of new and/or cryptic species. Sanger sequencing technology is the method of choice for obtaining standard 650 bp cytochrome c oxidase subunit I (COI) barcodes. However, DNA degradation/fragmentation makes it difficult to obtain a full-length barcode from old specimens. Mini-barcodes of 130 bp from the standard barcode region have been shown to be effective for accurate identification in many animal groups and may be readily obtained from museum samples. Here we demonstrate the application of an alternative sequencing technology, the four-enzymes single-specimen pyrosequencing, in rapid, cost-effective mini-barcode analysis. We were able to generate sequences of up to 100 bp from mini-barcode fragments of COI in 135 fresh and 50 old Lepidoptera specimens (ranging from 53–97 year-old). The sequences obtained using pyrosequencing were of high quality and we were able to robustly match all the tested pyro-sequenced samples to their respective Sanger-sequenced standard barcode sequences, where available. Simplicity of the protocol and instrumentation coupled with higher speed and lower cost per sequence than Sanger sequencing makes this approach potentially useful in efforts to link standard barcode sequences from unidentified specimens to known museum specimens with only short DNA fragments.

Validation and Development of COI Metabarcoding Primers for Freshwater Macroinvertebrate Bioassessment

“…However, the COI barcoding gene region shows high codon degeneracy throughout its sequence, making the design of such "truly" universal primers difficult (Deagle et al, 2014;Sharma and Kobayashi, 2014). Several COI barcoding primers with different levels of base degeneracy have been developed of which many are now used or could be suitable for metabarcoding studies (Figure 1, e.g., Folmer et al, 1994;Hebert et al, 2004;Meusnier et al, 2008;Van Houdt et al, 2010;Shokralla et al, 2011Shokralla et al, , 2015Zeale et al, 2011;Geller et al, 2013;Leray et al, 2013;Gibson et al, 2014;Brandon-Mong et al, 2015). However, often these primers were developed for a specific taxonomic group, purpose or ecosystem, for example the primers by Zeale et al (2011) which were originally developed for gut content analysis on bats but are now more widely used.…”

Species identification of bivalve molluscs by pyrosequencing

“…Even though the DNA‐based biomolecular techniques are more sophisticated than those based on protein patterns, the large amount of information that the DNA provides, as well as its stability, makes it the most suitable target for species identification . One of the most recent approaches used for identifying different species is the pyrosequencing method, which enables a faster bioluminometric real‐time sequence determination in solution . The pyrosequencing technology ‘sequencing by synthesis’ is based on a cascade of enzymatic reactions that leads to the production of light by the enzyme luciferase, and the amount of light is proportional to the number of incorporated nucleotides.…”

“…The pyrosequencing technology ‘sequencing by synthesis’ is based on a cascade of enzymatic reactions that leads to the production of light by the enzyme luciferase, and the amount of light is proportional to the number of incorporated nucleotides. Using this technique, a short stretch of 30–40 nucleotides downstream from the sequencing primer can be sequenced with a high degree of efficiency and accuracy . In this work, we developed two distinct protocols (later referred to as ‘16S protocol’ and ‘COI protocol’), capable of identifying in a rapid and univocal way different bivalve species commonly involved in commercial fraud in the Italian market from processed and/or unprocessed products.…”

PyroClean: Denoising Pyrosequences from Protein-Coding Amplicons for the Recovery of Interspecific and Intraspecific Genetic Variation

“…Like nuclear rRNA genes, mitochondrial 12S and 16S genes satisfy this criterion by the presence of highly conserved regions that span more variable regions. However, the evolutionary properties of protein-coding genes within the mitochondrial genome also provide the potential for universal cross-taxon amplification [16], and several recent studies demonstrate the potential for mtDNA COI primers to capture sample diversity for pyrosequencing [15], [17], [18]. Recently developed bioinformatic tools [19], [20] facilitate the design of degenerate primer pairs for improved cross-taxon amplification (e.g.…”