Computational protocol: Haemosporida prevalence and diversity are similar in endangered wild whooping cranes (Grus americana) and sympatric sandhill cranes (Grus canadensis)

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Protocol publication

[…] Amplicons were purified using ExoSAP-IT (Affymetrix, Santa Clara, CA) according to manufacturer's instructions. Purified samples were submitted for bi-directional sequencing to Eton Bioscience Inc. (San Diego, CA). Chromatographs were examined manually for quality and to assess congruence of forward and reverse strands using Clustal W within Mega 6·0 (Tamura et al. ). Sequences with double nucleotide peaks were separated using phasing. In this process, sequences with double nucleotide peaks were manually separated into all possible combinations of nucleotides that could have resulted in both nucleotides at a single polymorphic site. The resulting sequences were retained for analysis to determine the likely strains represented in the sample, however sequences with double nucleotide peaks were excluded from phylogenetic analysis. Sequences generated in this study were compared with known Haemosporida sequences using the BLAST tool in GenBank and were aligned with the closest matches and additional publicly available avian Haemosporida species sequences representative of unique clades in previous studies (Perkins & Schall, ; Martinsen et al. ; Outlaw & Ricklefs, ). Samples were considered positive for prevalence estimates if a DNA sequence was obtained for which the identity matched most closely to a Haemosporida species in GenBank. Due to the sequence homology in the cytb gene across Leucocytozoon, Plasmodium and Haemoproteus species, the PCR assay used to detect Leucocytozoon has been shown to produce false positive results for Leucocytozoon due to the presence of Plasmodium or Haemoproteus (Szollosi et al. ). Accordingly, we considered a sample positive for Leucocytozoon infection only if a DNA sequence was obtained for which the identity matched most closely to a Leucocytozoon species in GenBank.Phylogenetic relationships were analysed in Mega 6·0 using the maximum-likelihood method based on a general time reversible with gamma distribution (GTR+G) model of evolution using the bootstrap method with 1000 replicates (Hall, ). The model was selected based on fit estimated by the Akaike information criterion (AICc) and Bayesian information criterion (BIC). Each unique sequence produced during this project and utilized in the phylogenetic analysis was deposited in GenBank (Accession no. KX223847–KX223877); when sequences occurred more than once in the dataset, the accompanying notes in GenBank identify all samples that shared the same sequence. […]

Pipeline specifications

Software tools Clustal W, MEGA
Application Phylogenetics
Organisms Grus americana
Diseases Infection