Computational protocol: Lyme Disease, Virginia, USA, 2000–2011

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Protocol publication

[…] To extract total DNA, individual ticks were dried and flash-frozen by using liquid nitrogen, crushed by using a sterilized pestle, and processed with Qiagen DNeasy Blood and Animal Tissue Kit (QIAGEN, Valencia, CA, USA) by using manufacturer’s protocols. We tested for B. burgdorferi DNA by PCR amplification of the outer surface protein C (ospC) gene and the intergenic spacer region of 16S–23S rRNA genes (). Presence of amplified DNA was determined by gel electrophoresis, and samples that produced amplicons were purified with a QIAquick PCR Purification Kit (QIAGEN) and submitted for sequencing at the Nucleic Acids Research Facility at Virginia Commonwealth University (Richmond, VA, USA). We also performed PCR to amplify and subsequently sequence an ≈460-bp portion of the I. scapularis 16S rRNA gene using primers 16S +1 and 16S –1 (). Bidirectional chromatograms from all sequence data were assembled and initially analyzed with Sequencher 4.10.1 (Gene Codes, Ann Arbor, MI, USA). B. burgdorferi sequences were blasted by using GenBank (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to confirm species identification. Sequence data from I. scapularis 16S samples were aligned with reference sequences () by using ClustalW (http://www.clustal.org) implemented in MEGA 5.0 (http://www.megasoftware.net/), which was also used to select among models of evolution and to reconstruct phylogeny. […]

Pipeline specifications

Software tools Sequencher, Clustal W, MEGA
Application Phylogenetics
Organisms Borreliella burgdorferi, Ixodes scapularis, Homo sapiens
Diseases Lyme Disease