Computational protocol: DNA Barcoding in Pencilfishes (Lebiasinidae: Nannostomus) Reveals Cryptic Diversity across the Brazilian Amazon

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[…] Genomic DNA was isolated from muscle tissue based on standard techniques and protocols []. The partial mitochondrial COI gene (648pb) was amplified by PCR using a combination of primers FishF1, FishR1, FishF2 FishR2) [] or cock-tail C_VF1LF_t1- C_VR1LR_t1 under conditions previously described [].Polymerase chain reactions were performed in a total volume of 15μL (∼10–50 ng DNA template, 1X buffer (750 mM Tris-HCl, pH 8.8, 200 mM (NH4)2SO4), 1U Taq polymerase (Thermo Scientific, Waltham, USA), 0.2 mM dNTPs, 0.2 μM of each primer, 2 mM MgCl2, and ultrapure water. PCR cycling was performed with the initial denaturation for 2 min at 95°C followed by 35 cycles of 30s at 95ºC, 30s at 52ºC-54ºC, 1 min at 72ºC and with a final extension for 10 min at 72ºC. PCR products were resolved on 1% agarose gels and purified using polyethylene glycol 8000 (USB, Cleveland, USA). The bi-directional sequencing was performed utilizing an ABI BigDye TM Terminator v.3.1 Cycle Sequencing Ready Reaction Kit and an ABI 3130xl DNA Analyzer (Applied Biosystems, Foster City, USA).Data sequences, collection sites, primers details and trace files were submitted to the Barcode of Life database (BOLD; http//www.boldsystems.org) in under project “Barcoding of Lebiasinids.” [...] Consensus sequences for the COI gene were generated using the BioEdit program [], and after editing the sequences, the final matrix was 574bp.All sequences were analyzed using MEGA 5 to check the occurrence of deletions, insertions, and stop codons. Search tools with local alignment were used to identify the sequence in GenBank and the BOLD. Sequences were aligned using Clustal W [], and the program DnaSP version 4.0 [] was used to determine the nucleotide composition, number of polymorphic sites, and haplotypes diversity.The genetic distance among and within observed clusters was calculated using the Kimura-2-parameter (K2P) model. A Bayesian phylogenetic analysis was conducted using MrBayes 3.2 []. For this analysis, Markov chain Monte-Carlo sampling was conducted every 120,000 generations until the standard deviation of split frequencies was <0.01. A burn-in period equivalent to 25% of the total generations was necessary to recapitulate the parameter values and trees. The parameter values were evaluated based on 95% credibility levels to ensure a sufficient number of generations had been run for the analysis. A neighbor-joining (NJ) tree of K2P distances was created to provide a graphic representation of the relationships among specimens and clusters with MEGA 6.0 []. Bootstrap resampling [] was applied to assess the support for individual nodes using 1000 pseudo-replicates. The program Haploviewer[] was used to construct a tree-based haplotype network. Independent networks were regarded as unconfirmed candidate species. […]

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