Computational protocol: Evidence for an African Cluster of Human Head and Body Lice with Variable Colors and Interbreeding of Lice between Continents

Similar protocols

Protocol publication

[…] In this study, we had four parameters to compare: ecology, geographical origin, phylogeny based on 4 spacers and color. So we decided to sequence only Clade A lice because this is the only clade that comprise both head and body lice and therefore is a specific problem of public health as body lice are vectors of outbreaks. Lice from four countries were genotyped with the MST method including head lice from Senegal collected in October and November 2010 , body lice from Southern Ethiopia collected in 2010 , body lice from Rwanda collected in 2011 (three lice per individual) and both head and body lice from Burundi collected in 2008 (three lice of each ecotype per individual). Altogether, 19 Clade A lice were used in this study (). All lice were stored at −20°C before DNA extraction with the QiAamp Tissue kit (QIAGEN, Hilden, Germany). Senegalese and Rwandan lice were photographed with a camera (Olympus DP71) fixed on a low power stereo microscope (Olympus SZX16). The lice from Ethiopia and France were photographed in the field (with a Nikon D90) either in the hand of one of the authors (Ethiopia) or directly on the clothes of the infested person (France). Four intergenic spacers (S2, S5, PM1, PM2) known to be very polymorphic were used in this study. The sequencing was performed following the protocol previously described with some minor modifications. Briefly, the PCR reactions were prepared on ice and contained 3 µl of the DNA template, 4 µl of Phusion HF Buffer, 250 µM of each nucleotide, 0.5 µM of each primer, 0.2 µl of Phusion DNA Polymerase (Ozyme) and water to a final reaction mixture volume of 20 µl. The reactions were performed in a PTC-200 automated thermal cycler (MJ research, Waltham, MA, USA). The cycling conditions were 98°C for 30 sec; 35 cycles of 5 sec at 98°C, 30 sec at 56°C, 15 sec at 72°C; and a final extension time of 5 min at 72°C. The success of the PCR amplification was then verified by migration of the PCR product on an agarose gel. The NucleoFast 96 PCR Plates (Macherey-Nagel EURL, France) and BigDye Terminator version 1.1 cycle sequencing-ready reaction mix (Applied Biosystems, Foster City, CA) were then used to purify the PCR products before sequencing in both directions with the same primers used in the PCR amplification. The ABI 3100 automated sequencer (Applied Biosystems) resolved the sequenced products. The program ChromasPro was used to analyze, assemble and correct the sequences. When forward and reverse sequences could not be assembled, they were analyzed separately and resolved. Each sequence was aligned with genotypes published in Genbank ) for identification. When less than 100% homology was obtained, the new genotype was recorded, a new number was assigned to it and sequences were deposited in Genbank under accession numbers from JQ652371 and JQ652455. When the chromatogram indicated possible heterozygotic sequences, these samples were cloned to identify the different allelic sequences. The PCR products were cloned into pGEM-T-Easy vector (Promega, Madison, WI) following the manufacturer's instructions with some minor modifications. Before ligation, A-overhangs were added to the PCR product. This was performed by incubating 4.2 µl of purified PCR product with 1 U of DyNAzyme II DNA polymerase, 0.5 µl of Optimized DyNAzyme Buffer and 0.2 mM dATP with a final volume of 5 µl for 20 min at 72°C. Then, each reaction was ligated with 5 µl of 2X Rapid Ligation Buffer, 3 µl of purified A-overhangs-PCR product, 1 µl of T4 DNA ligase and 1 µl of pGEM®-T Easy Vector and incubated overnight at 15°C. Each ligation reaction was transformed into 50 µl of JM109 High Efficiency Competent Cells by letting them incubate together on ice for 20 min before a 1 min heat shock in a 42°C water bath. 950 µl of LB broth was then added to cells before incubation on a 37°C shaker for 1.5 hours. 300 µl of transformed cells was plated onto LBagar/ampicillin/IPTG/X-Gal plates and these were incubated overnight at 37°C. Eight white colonies per sample were then resuspended in 100 µl of RNase/DNase free water and subsequently PCR amplified and sequenced using the M13 universal and M13 reverse primers. Phylogenetic analysis was done using our data and data from a previous study performed in 2010, which included lice from France, Portugal, Mexico, Russia, Burundi and Rwanda (these lice were given names beginning with “li-” in the trees) . Two phylogenetic methods, Maximum Parsimony (MP) and Maximum Likelihood (ML), were used to infer the trees for each individual spacer. For each spacer, the louse nucleic sequences were aligned with the genotype 1 found in Genbank (EU928781.1, EU928804.1, EU913096.1, EU913178.1 for PM1, PM2, S2 and S5, respectively) using the MUSCLE algorithm . Then, trees were drawn within the MEGA 5 software with complete deletion . A tree was also constructed with the concatenated sequences of the four spacers (S2, S5, PM1, PM2). Because the louse genome is diploid, in instances where there were two different alleles per locus, all possibilities of concatenation were made for each louse and all of them were taken into account in the tree (the possibilities were labeled from a to h in the tree). […]

Pipeline specifications

Software tools ChromasPro, MUSCLE, MEGA
Applications Phylogenetics, Nucleotide sequence alignment
Organisms Homo sapiens, Pediculus humanus
Diseases Lice Infestations