Computational protocol: Molecular characterization of cryptic and sympatric lymnaeid species from the Galba/Fossaria group in Mendoza Province, Northern Patagonia, Argentina

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Protocol publication

[…] Genomic DNA was extracted from the head-foot tissue (preserved in 100% ethanol, as described above) of 143 individual snails using a standard CTAB protocol []. Separate PCRs were performed to amplify fragments of the cytochrome oxidase sub-unit 1 (COI) gene and the 16S ribosomal RNA gene (16S), using standard universal primers (LCO1490 and HCO2198 and 16arm/16brm) and cycling conditions [,]. Amplifications were done using Promega Go-Taq (Promega Corporation, Madison, UK) in a 25 μl reaction. All successful amplifications were purified using either a Millipore PCR96 Cleanup kits on a vacuum manifold (Millipore, Billerica, USA) or ExoSAP-IT (GE Healthcare Biosciences, Pittsburgh, USA) as per manufacturer’s instructions, using pure water for washing and resuspension. Product concentration was quantified on a Nanodrop ND-1000 Spectrophotometer (Nanodrop Technologies Inc., Willington, USA), and sequencing reactions were performed on purified PCR products using an Applied Biosystems Big Dye Kit (version 1.1) and run on an Applied Biosystems 3730 DNA Analyzer (Applied Biosystems, Carlsbad, USA). Sequences were assembled and edited by eye using Sequencher v 4.8 (Gene Codes Corporation, Ann Arbor, Michigan, USA: http://www.genecodes.com) and Geneious v 5.3.6 (Biomatters Ltd., Auckland, New Zealand) and aligned using MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/). [...] Overall, 143 snails from the seven collection sites were sequenced for the COI gene, and 49 were sequenced for the 16S gene. The mean corrected genetic distances were calculated for within and between each haplotype as well as each species. The best-fit model of nucleotide substitution for each sequence dataset was determined by comparing model likelihoods based on neighbour-joining trees, using PAUP version 4 [].Maximum likelihood-based tree-building was used to investigate the relationships between the mitochondrial haplotypes, using the same model of nucleotide substitution as for the corrected distance estimations. Node support was determined through bootstrapping (1000 replicates in all cases). PhyML [] was used to create the maximum likelihood tree and subsequent bootstrapping. Trees were visualised and edited using FigTree v 1.3.1 (http://tree.bio.ed.ac.uk/software/figtree/). […]

Pipeline specifications

Software tools Sequencher, Geneious, MUSCLE, PAUP*, PhyML, FigTree
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Bos taurus
Diseases Fascioliasis, Liver Diseases, Trematode Infections