Computational protocol: Performance of Glutamate Dehydrogenase and Triose Phosphate Isomerase Genes in the Analysis of Genotypic Variability of Isolates of Giardia duodenalis from Livestocks

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Protocol publication

[…] Positive nested-PCR products were purified with Sephacryl 400 resin (Ilustra-MicroSpin S400 HR Columns) and sequenced in a single direction. Reactions were performed in a Mastercycler pro thermocycler (Eppendorf, Brazil) using Big Dye terminator V.3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA). Products were read using the automatic sequencer ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).The quality of the partial sequences and the clustering of the fragments were obtained with the use of Sequence Scanner version 1.0 (Copyright Applied Biosystems, Foster City, CA, USA). Nucleotide alignment was performed manually using BioEdit Sequence Alignment Editor (Hall, 1999) and using as a base the homologous sequences available on GenBank: M84604 (assemblage A), AY826193 (assemblage B), U60982 (assemblage C), U60986 (assemblage D), AY178741 (assemblage E), and AF069057 (assemblage F) for gdh gene and AY655704 (assemblage A, sub-assemblage AI), U57897 (assemblage A, sub-assemblage AII), AF069561 (assemblage B, sub-assemblage BIII), AF069560 (assemblage B, sub-assemblage BIV), AY228641 (assemblage C), DQ246216 (assemblage D), AY228645 (assemblage E), and AF069558 (assemblage F) for tpi gene.In order to determine the phylogenetic relationship among assemblages, phylograms were constructed using Mega v.5.1 Beta, with the neighbor-joining method, with bootstrap values established in 1000 replicates. […]

Pipeline specifications

Software tools BioEdit, MEGA-V
Applications Phylogenetics, GWAS
Organisms Giardia intestinalis, Sus scrofa, Ovis aries, Bos taurus