An In silico approach for the evaluation of DNA barcodes

By Gentile Francesco Ficetola, É. Coissac, S. Zundel, Tiayyba Riaz, W. Shehzad, J. Bessière, P. Taberlet, F. Pompanon. BMC Genomics, 2010

469 citations recorded by the source corpus.

BackgroundDNA barcoding is a key tool for assessing biodiversity in both taxonomic and environmental studies. Essential features of barcodes include their applicability to a wide spectrum of taxa and their ability to identify even closely related species. Several DNA regions have been proposed as barcodes and the region selected strongly influences the output of a study. However, formal comparisons between barcodes remained limited until now. Here we present a standard method for evaluating barcode quality, based on the use of a new bioinformatic tool that performs in silico PCR over large databases. We illustrate this approach by comparing the taxonomic coverage and the resolution of several DNA regions already proposed for the barcoding of vertebrates. To assess the relationship between in silico and in vitro PCR, we also developed specific primers amplifying different species of Felidae, and we tested them using both kinds of PCRResultsTests on specific primers confirmed the correspondence between in silico and in vitro PCR. Nevertheless, results of in silico and in vitro PCRs can be somehow different, also because tuning PCR conditions can increase the performance of primers with limited taxonomic coverage. The in silico evaluation of DNA barcodes showed a strong variation of taxonomic coverage (i.e., universality): barcodes based on highly degenerated primers and those corresponding to the conserved region of the Cyt-b showed the highest coverage. As expected, longer barcodes had a better resolution than shorter ones, which are however more convenient for ecological studies analysing environmental samples.ConclusionsIn silico PCR could be used to improve the performance of a study, by allowing the preliminary comparison of several DNA regions in order to identify the most appropriate barcode depending on the study aims.

Subfields: Ecology, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/df17443c6dde654fb85996aac5e263d10611cdad

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Citations in the corpus

REVIEW: The detection of aquatic animal species using environmental DNA – a review of eDNA as a survey tool in ecology (2014, methodology): “Primer and probe sequences have been validated by careful design either manually or by the use of available software such as Primer 3 (Rozen & Skaletsky 2000), Primer Express (Gene Codes Corporation, Ann Arbor, MI, USA) or ecoPCR (Ficetola et al. 2010) followed by”
Towards next‐generation biodiversity assessment using DNA metabarcoding (2012, methodology): “2011) and for testing their characteristics (ecoPCR program; Bellemain et al. 2010; Ficetola et al. 2010).”
obitools: a unix‐inspired software package for DNA metabarcoding (2016, methodology): “The OBITOOLS have already been used in many published studies (e.g. Ficetola et al. 2010; Kowalczyk et al. 2011; Yoccoz et al. 2012; Bellemain et al. 2013), demonstrating their usefulness for various applications related to metabarcoding.”
Metabarcoding: A powerful tool to investigate microbial communities and shape future plant protection strategies (2017, methodology): “Software programs such as ecoPrimers (Riaz et al., 2011), ecoPCR (Ficetola et al., 2010), and PrimerProspector (Walters et al., 2011) can greatly assist in the design of barcode primers.”
A comprehensive and comparative evaluation of primers for metabarcoding eDNA from fish (2020, methodology): “…the amplification coverage and taxonomic resolution of the fish metabarcoding primers using in silico PCR implemented in the ECOPCR program (Ficetola et al., 2010) with the following settings: (a) amplicon length in the range of 15‒500 bp (excluding primers) and (b) a maximum of three…”
Lake sediment multi-taxon DNA from North Greenland records early post-glacial appearance of vascular plants and accurately tracks environmental changes (2015, methodology): “In addition, reference libraries for each organism group were created with each of the primer pairs from the EMBL Nucleotide Database standard release 113 by extracting sequences of the targeted region using ecoPCR (Ficetola et al., 2010).”
ITS as an environmental DNA barcode for fungi: an in silico approach reveals potential PCR biases (2010, methodology): “For these purposes we performed in silico PCR using various primer combinations on target sequences retrieved from EMBL databases as well as subset databases using the bioinformatic tool EcoPCR [ 23 ].”
Species-specific detection and quantification of environmental DNA from marine fishes in the Baltic Sea (2019, background): “, 2016) and ecoPCR (Bellemain et al., 2010; Ficetola et al., 2010) at a generic level, with data analysis in R v.”
Persistence of Environmental DNA in Freshwater Ecosystems (2011, background): “other species were not amplified at the same time as the species of interest) [30].”
Who is eating what: diet assessment using next generation sequencing (2012, methodology): “About 60% of the taxa potentially amplified differ at each priming site by 4–8 mismatches, probably leading to a failure or a low rate of amplification (Ficetola et al. 2010).”
Next‐generation monitoring of aquatic biodiversity using environmental DNA metabarcoding (2016, methodology): “The PCR products were purified and sequenced (forward and reverse) using Sanger technology at the Eurofins MWG Operon sequencing facilities (Ebersberg, Germany).”
Non‐specific amplification compromises environmental DNA metabarcoding with COI (2019, methodology): “Discrimination as a proportion of the total number of species was calculated following Ficetola et al. (2010): ‘A taxon unambiguously TA B L E 1 Primer sets assessed in this study.”
A new versatile primer set targeting a short fragment of the mitochondrial COI region for metabarcoding metazoan diversity: application for characterizing coral reef fish gut contents (2013, background): “A single attempt was made at designing a versatile primer to amplify a shorter “mini-barcode” COI region [17], but it has received limited use due to large numbers of mismatches in the priming site that affects its efficiency across a broad range of taxa [33].”