Computational protocol: Demographic Variation of Wolbachia Infection in the Endangered Mitchell’s Satyr Butterfly

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Protocol publication

[…] Samples that tested positive for PCR amplification of Wspec were cleaned with ExoSAP-IT (Thermo Fisher Scientific, Waltham, MA, USA) to remove excess nucleotides. Forward and reverse cycle sequencing reactions were run separately using BigDye Terminators (Applied Biosystems) and the Wspec primers. Dye terminator removal and sequencing was conducted at Georgia Genomics Institute. Chromatogram files for each sequence were cleaned by removing low quality and aligned using Sequencher version 5.5.1 (Gene Codes, Ann Arbor, MI, USA). Consensus sequences were generated from the forward and reverse sequences for each sample. To confirm the amplified 16s fragments were from Wolbachia, the consensus sequences were used to conduct TBLASTX [] sequence similarity comparisons against the National Center for Biotechnology Information (NCBI) nucleotide database restricted to sequences from (i) bacteria only and (ii) Wolbachia pipientis only. The sequence for the 16s fragment isolated from N. mitchellii and M. cymela is available at NCBI genbank (Accession # MF002138 and MF002139). […]

Pipeline specifications

Software tools Sequencher, TBLASTX
Application Nucleotide sequence alignment
Diseases Bacterial Infections, Infection