Verhey, Theodore BCastellanos, MildredChaconas, George2018-12-072018-12-072018-01Verhey, T. B., Castellanos, M., & Chaconas, G. (2018). Analysis of recombinational switching at the antigenic variation locus of the Lyme spirochete using a novel PacBio sequencing pipeline. Molecular microbiology, 107(1), 104-115.29105221http://hdl.handle.net/1880/109290https://doi.org/10.11575/PRISM/43815The Lyme disease spirochete evades the host immune system by combinatorial variation of VlsE, a surface antigen. Antigenic variation occurs via segmental gene conversion from contiguous silent cassettes into the vlsE locus. Because of the high degree of similarity between switch variants and the size of vlsE, short-read NGS technologies have been unsuitable for sequencing vlsE populations. Here we use PacBio sequencing technology coupled with the first fully-automated software pipeline (VAST) to accurately process NGS data by minimizing error frequency, eliminating heteroduplex errors and accurately aligning switch variants. We extend earlier studies by showing use of almost all of the vlsE SNP repertoire. In different tissues of the same mouse, 99.6% of the variants were unique, suggesting that dissemination of Borrelia burgdorferi is predominantly unidirectional with little tissue-to-tissue hematogenous dissemination. We also observed a similar number of variants in SCID and wild-type mice, a heatmap of location and frequency of amino acid changes on the 3D structure and note differences observed in SCID versus wild type mice that hint at possible amino acid function. Our observed selection against diversification of residues at the dimer interface in wild-type mice strongly suggests that dimerization is required for in vivo functionality of vlsE.enjournal articlehttp://dx.doi.org/10.1111/mmi.13873