Abstract
The process of molecular identification of species known as DNA barcoding consists on discriminating taxa based on cumulative differences of their DNA sequences and is widely used within animals, including birds. Finding the best genomic tools, such as primers, to reach most of the species within the studied groups and evaluating the coverage breadth and physicochemical properties of primers before testing in the laboratory, can save time and financial resources. Therefore, this work aimed to retrieve the available primers for the COI gene currently used on the molecular identification of birds and evaluate them for its coverage range, physicochemical properties, and performance on in silico PCR. Afterwards, we provide the best primer subsets to cover the highest number of avian sequences and the best individual primers for each bird order in terms of coverage breadth. Thirty-one bird orders had at least one COI sequence to serve as template, 152 primers available for assessing the COI gene were evaluated, and 118 could bind to at least one template sequence. No primer subset alone could cover all template sequences; however, when combining two subsets, the complete coverage of avian COI sequences analyzed was achieved. We were able to find optimal primer subsets for more specific taxonomic groups or for analysis involving the complete avian group, in order to guide researchers in the process of choosing the best primer set for each individual barcoding goal.
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The primers’ sequences analyzed in this current study were retrieved from previously published papers indexed in the Web of Science Database available at https://clarivate.com/webofsciencegroup/solutions/web-of-science/ (see Supplementary Table 1 for further reference information). The mitochondrial genome sequences datasets analyzed in this study were retrieved from the Nucleotide database from the GenBank sequence archive of the National Center for Biotechnology Information (NCBI), available at https://www.ncbi.nlm.nih.gov/ (see Supplementary Table 3 for GenBank Accession numbers).
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Acknowledgements
We would like to thank the developer Marcelo Amoretti for all the technical support granted on the setup and operation of the data processing tools used on this work. And finally, we thank the “openPrimeR” developer, Matt Doering, for kindly answering the operational questions regarding his software that made this work viable.
Funding
Our current research in Genetics and Genomics is developed in the context of National Institutes for Science and Technology (INCT) in Ecology, Evolution and Biodiversity Conservation, supported by MCTIC/CNPq (proc. 465610/2014–5), FAPEG (Fundação de Amparo à Pesquisa do Estado de Goiás), and PRONEX/FAPEG/CNPqN° 06/2016. Work by A.A.M was supported by a fellowship from CAPES (Coordenação de Aperfeiçoamento de Pessoal de Nível Superior). Work by R.N. was supported by a fellowship from CNPq (Conselho Nacional de Desenvolvimento Científico e Tecnológico). Work by M.P.C.T. has been continuously supported by productivity fellowships from CNPq.
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de Melo, A.A., Nunes, R. & Telles, M.P. Same information, new applications: revisiting primers for the avian COI gene and improving DNA barcoding identification. Org Divers Evol 21, 599–614 (2021). https://doi.org/10.1007/s13127-021-00507-x
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DOI: https://doi.org/10.1007/s13127-021-00507-x