Computational protocol: Genotypic diversity of merozoite surface antigen 1 of Babesia bovis within an endemic population

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Protocol publication

[…] Parasite DNA was amplified with oligonucleotide primers msa-1 forward (5′-ATGGCTACGTTTGCTCTTTTCATTTC-3′) and msa-1 reverse (5′-AAATGCAGAGAGAACGAAGTAGC-3′) designed from the Texas T2Bo isolate of B. bovis (XP_001608956.1). These two primers anneal to highly conserved 5′ and 3′ terminal sequences of msa-1 . PCR conditions were 95 °C for 3 min, followed by 35 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 90 s, and a final extension of 72 °C for 7 min. B. bovis Mo7 biological clone DNA and water were used as positive and negative amplification controls, respectively. Proof-reading Taq polymerase (Invitrogen) was used throughout all reactions. PCR was set up in triplicate and the resulting amplicons were cloned into pCR4-TOPO cloning vector (Invitrogen). Plasmid DNA from 30 clones from each sample was extracted using QIAprep® Spin Miniprep Kit (Qiagen) and sequenced with M13 forward (5′-GTAAAACGACGGCCAG-3′) and M13 reverse (5′-CAGGAAACAGCTATGAC-3′) primers using standard ABI chemistries. All colonies extracted from each sample were sequenced to ensure that variant MSA-1 products, if present, could be detected. All preliminary sequence analyses performed in this study were conducted at the nucleotide and amino acid levels; however, determination of genotypes and subsequent distributions within the study cohort were conducted at the amino acid level only since many of the single nucleotide polymorphisms observed were synonymous. Overall distribution of genotypes was determined based on the initial sequences/sample (). The proportion of genotypes detected in each sample was not used in the analysis, only their presence. Additional sequencing of some samples was carried out to confirm that genotypes obtained during the first round of sequencing were reproducible and that no minor genotypes were detected the second time around. Power calculations were performed using the binomial theorem to estimate probability of detecting an individual parasite genotype. Thirty clones per sample provided a 95% probability of detecting a genotype present in the population at a level as low as 9.5%. Statistical significance of genotype changes within an animal between the two sampling periods was calculated using binomial test for equal proportions with p ≤ 0.05. To determine potential sequence variability of MSA-1 in vitro, 500 ng of gDNA from three separate batches of established long-term blood cultured B. bovis T2Bo strain was used to amplify msa-1. Thirty-three clones were selected and sequenced as described above. Analysis of sequence data of MSA-1, including phylogeny and ranges of amino acid identities within and between cluster groups were determined using MacVector® with Assembler, Version 10.0. Multiple amino acid alignments were performed using ClustalW as the default setting on MacVector and Neighbor joining tree analysis was predicted using Bootstrap with random tie breaking as a feature. Poisson distribution that would measure the distances between nodes and gap sites was ignored. […]

Pipeline specifications

Software tools MacVector, Clustal W
Application Phylogenetics
Organisms Babesia bovis
Diseases Babesiosis, Coinfection