High, clustered, nucleotide diversity in the genome of Anopheles gambiae revealed through pooled-template sequencing: implications for high-throughput genotyping protocols

<p>Abstract</p> <p>Background</p> <p>Association mapping approaches are dependent upon discovery and validation of single nucleotide polymorphisms (SNPs). To further association studies in <it>Anopheles gambiae </it>we conducted a major resequencing programme, primarily targeting regions within or close to candidate genes for insecticide resistance.</p> <p>Results</p> <p>Using two pools of mosquito template DNA we sequenced over 300 kbp across 660 distinct amplicons of the <it>An. gambiae </it>genome. Comparison of SNPs identified from pooled templates with those from individual sequences revealed a very low false positive rate. False negative rates were much higher and mostly resulted from SNPs with a low minor allele frequency. Pooled-template sequencing also provided good estimates of SNP allele frequencies. Allele frequency estimation success, along with false positive and negative call rates, improved significantly when using a qualitative measure of SNP call quality. We identified a total of 7062 polymorphic features comprising 6995 SNPs and 67 indels, with, on average, a SNP every 34 bp; a high rate of polymorphism that is comparable to other studies of mosquitoes. SNPs were significantly more frequent in members of the cytochrome p450 mono-oxygenases and carboxy/cholinesterase gene-families than in glutathione-S-transferases, other detoxification genes, and control genomic regions. Polymorphic sites showed a significantly clustered distribution, but the degree of SNP clustering (independent of SNP frequency) did not vary among gene families, suggesting that clustering of polymorphisms is a general property of the <it>An. gambiae </it>genome.</p> <p>Conclusion</p> <p>The high frequency and clustering of SNPs has important ramifications for the design of high-throughput genotyping assays based on allele specific primer extension or probe hybridisation. We illustrate these issues in the context of the design of Illumina GoldenGate assays.</p

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Thesis (M.S.)--Massachusetts Institute of Technology, Dept. of Mechanical Engineering, 1968.Bibliography: leaves 88-92.M.S

Adaptive potential of hybridization among malaria vectors: Introgression at the immune locus TEP1 between Anopheles coluzzii and A. gambiae in 'Far-West' Africa

“Far-West” Africa is known to be a secondary contact zone between the two major malaria vectors Anopheles coluzzii and A. gambiae.We investigated gene-flow and potentially adaptive introgression between these species along a west-to-east transect in Guinea Bissau, the putative core of this hybrid zone. To evaluate the extent and direction of gene flow, we genotyped site 702 in Intron-1 of the para Voltage-Gated SodiumChannel gene, a species-diagnostic nucleotide position throughout most of A. coluzzii and A. gambiae sympatric range. We also analyzed polymorphismin the thioester-binding domain (TED) of the innate immunity-linked thioester-containing protein 1 (TEP1) to investigate whether elevated hybridization might facilitate the exchange of variants linked to adaptive immunity and Plasmodium refractoriness. Our results confirm asymmetric introgression of genetic material from A. coluzzii to A. gambiae and disruption of linkage between the centromeric "genomic islands" of inter-specific divergence. We report that A. gambiae from the Guinean hybrid zone possesses an introgressed TEP1 resistant allelic class, found exclusively in A. coluzzii elsewhere and apparently swept to fixation inWest Africa (i.e. Mali and Burkina Faso). However, no detectable fixation of this allele was found in Guinea Bissau, which may suggest that ecological pressures driving segregation between the two species in larval habitats in this region may be different from those experienced in northern and more arid parts of the species’ range. Finally, our results also suggest a genetic subdivision between coastal and inland A. gambiae Guinean populations and provide clues on the importance of ecological factors in intra-specific differentiation processes

Detection of Tyrosinase Autoantibodies in Patients With Vitiligo Using 35S-Labeled Recombinant Human Tyrosinase in a Radioimmunoassay

Tyrosinase antibodies recently have been reported to occur frequently in patients with vitligo. We describe the detection of tyrosinase antibodies in vitiligo patients using in vitro 35S-labeled human tyrosinase in a radioimmunoassay. Of 46 vitiligo sera examined in the assay, five (10.9%) were found to be positive for tyrosinase antibodies. In contrast, 20 control sera and sera from 10 patients with Hashimoto's thyroiditis were negative. Four of the sera positive in the radioimmunoassay were also positive in an ELISA using mushroom tyrosinase as antigen. Absorption studies indicated that pre-incubation with mushroom tyrosinase absorbed out the immunoreactivity of the positive sera in the radioimmunoassay, suggesting cross-reactivity, but this absorption was never complete, indicating that there are tyrosinase antibodies in human sera that do not react with the mushroom protein. There was no obvious association between the presence of tyrosinase antibodies and the age of the patients (range: 22–62 y), their duration of disease (range: 5–20 y), or the type of vitiligo (one segmental, one symmetricallperiorificial, three symmetrical), although the three patients with the highest antibody levels also had an associated autoimmune disorder (one with Graves' disease; two with autoimmune hypothyroidism). The results confirm that tyrosinase autoantibodies are present in the sera of vitiligo patients but at a low frequency. The technique described is sensitive and quantitative and allows the detection of confirmational epitopes. It will be useful in longitudinal studies to determine the relation between the clinical features of vitiligo and tyrosinase antibody levels

Autoantigens in Vitiligo Identified by the Serological Selection of a Phage-Displayed Melanocyte cDNA Expression Library

Vitiligo is an acquired idiopathic hypomelanotic disorder characterized by circumscribed depigmented macules resulting from the loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, autoimmunity may play a role in the development of the disease. The present study was undertaken to investigate the applicability of phage display technology to identify B-cell autoantigens in vitiligo. A melanocyte cDNA phage display library was subjected to rounds of enrichment with vitiligo patient IgG. Subsequently, enriched IgG-binding peptides representing putative autoantigens were identified by sequencing their encoding cDNAs. Radioimmunoassays were used to confirm the immunoreactivity of vitiligo patient (n=61) and control (n=28) sera to several of the putative autoantigens. Non-segmental vitiligo patient sera (n=53) were positive for antibody (Ab) reactivity to gamma-enolase (8%); alpha-enolase (9%); heat-shock protein 90 (13%); osteopontin (4%); ubiquitin-conjugating enzyme (15%); translation-initiation factor 2 (6%); and GTP-binding protein, Rab38 (15%). Ab reactivity to at least one of the previously unknown autoantigens was detected in 51% of patients with non-segmental vitiligo. In contrast, Ab reactivity in a group of patients with segmental vitiligo (n=8) was not demonstrated. Overall, the study indicated that the targets of autoantibodies in vitiligo patients can be revealed by employing the methodology of phage display

Environmental requirements for three sea pen species: relevance to distribution and conservation

The aim is to determine the environmental requirements, estimate the extent of suitable habitat for three sea pen species, and assess the implications for marine protected areas (MPAs). The sea pen Funiculina quadrangularis and the habitat associated with two further sea pen species, Virgularia mirabilis and Pennatula phosphorea, are of key conservation importance and are recommended for protection within MPAs. This study models their potential distributions using the MAXimum ENTropy algorithm and assesses these in relation to five possible marine protected areas (pMPAs) proposed for Scottish waters. Metrics relevant to assessing the efficacy of MPAs are also presented. Four environmental variables of prime importance for predicting the presence of all three species of sea pen were identified: mud, minimum salinity, depth, and gravel. The habitat suitability index increased with mud content. The modelled distribution of F. quadrangularis indicated a deeper distribution than V. mirabilis or P. phosphorea and was not present in sediment with gravel content above 30%. Pennatula phosphorea had the smallest area of suitable habitat, while V. mirabilis had the largest. The percentage predicted suitable area for each species that was encompassed by the five pMPAs ranged from 11% for F. quadrangularis to 15% for P. phosphorea. Some of the largest areas predicted as suitable for F. quadrangularis lay outside the pMPAs. The model results indicated differences in the environmental requirements of the three species of sea pen that can be linked to the autecology of each species. Patch sizes, calculated from a binary output of the model, were used to estimate the degree of habitat fragmentation, thereby giving a partial assessment of the adequacy criterion for these pMPAs. The results suggest that potential MPAs within the study area cover sizeable areas of potential sea pen habitat. However, further areas suitable for F. quadrangularis could be considered

Plasmodium falciparum: linkage disequilibrium between loci in chromosomes 7 and 5 and chloroquine selective pressure in Northern Nigeria.

In view of the recent discovery (Molecular Cell 6, 861-871) of a (Lys76Thr) codon change in gene pfcrt on chromosome 7 which determines in vitro chloroquine resistance in Plasmodium falciparum, we have re-examined samples taken before treatment in our study in Zaria, Northern Nigeria (Parasitology, 119, 343-348). Drug resistance was present in 5/5 cases where the pfcrt 76Thr codon change was seen (100% positive predictive value). Drug sensitivity was found in 26/28 cases where the change was absent (93% negative predictive value). Allele pfcrt 76Thr showed strong linkage disequilibrium with pfmdr1 Tyr86 on chromosome 5, more complete than that between pfcrt and cg2 alleles situated between recombination cross-over points on chromosome 7. Physical linkage of cg2 with pfcrt may account for linkage disequilibrium between their alleles but in the case of genes pfmdr1 and pfcrt, on different chromosomes, it is likely that this is maintained epistatically through the selective pressure of chloroquine

Limited genomic divergence between intraspecific forms of Culex pipiens under different ecological pressures

Abstract Background: Divergent selection can be a major driver of ecological speciation. In insects of medical importance, understanding the speciation process is both of academic interest and public health importance. In the West Nile virus vector Culex pipiens, intraspecific pipiens and molestus forms vary in ecological and physiological traits. Populations of each form appear to share recent common ancestry but patterns of genetic differentiation across the genome remain unknown. Here, we undertook an AFLP genome scan on samples collected from both sympatric and allopatric populations from Europe and the USA to quantify the extent of genomic differentiation between the two forms. Results: The forms were clearly differentiated but each exhibited major population sub-structuring between continents. Divergence between pipiens and molestus forms from USA was higher than in both inter- and intra-continental comparisons with European samples. The proportion of outlier loci between pipiens and molestus (≈3 %) was low but consistent in both continents, and similar to those observed between sibling species of other mosquito species which exhibit contemporary gene flow. Only two of the outlier loci were shared between inter-form comparisons made within Europe and USA. Conclusion: This study supports the molestus and pipiens status as distinct evolutionary entities with low genomic divergence. The low number of shared divergent loci between continents suggests a relatively limited number of genomic regions determining key typological traits likely to be driving incipient speciation and/or adaptation of molestus to anthropogenic habitats