The rapid identification of antimicrobial resistance is essential for effective treatment of highly resistant Mycobacterium tuberculosis. Whole-genome sequencing provides comprehensive data on ...resistance mutations and strain typing for monitoring transmission, but unlike for conventional molecular tests, this has previously been achievable only from cultures of M. tuberculosis. Here we describe a method utilizing biotinylated RNA baits designed specifically for M. tuberculosis DNA to capture full M. tuberculosis genomes directly from infected sputum samples, allowing whole-genome sequencing without the requirement of culture. This was carried out on 24 smear-positive sputum samples, collected from the United Kingdom and Lithuania where a matched culture sample was available, and 2 samples that had failed to grow in culture. M. tuberculosis sequencing data were obtained directly from all 24 smear-positive culture-positive sputa, of which 20 were of high quality (>20× depth and >90% of the genome covered). Results were compared with those of conventional molecular and culture-based methods, and high levels of concordance between phenotypical resistance and predicted resistance based on genotype were observed. High-quality sequence data were obtained from one smear-positive culture-negative case. This study demonstrated for the first time the successful and accurate sequencing of M. tuberculosis genomes directly from uncultured sputa. Identification of known resistance mutations within a week of sample receipt offers the prospect for personalized rather than empirical treatment of drug-resistant tuberculosis, including the use of antimicrobial-sparing regimens, leading to improved outcomes.
Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African ...European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.
The Cape Floristic Region (CFR) is well-known for its floral diversity, yet also contains a rich herpetofauna with >180 species, 28% of which are endemic. Recent studies conducted on CFR lizards ...indicated that phylogeographic patterns show some congruency, and that the western CFR shows higher overall diversity in the form of population and/or clade turnover. Here, we combine mitochondrial sequence data from two published (
Bradypodion spp. and
Agama atra) and one new dataset (
Pedioplanis burchelli) to investigate whether geographic patterns of genetic diversity could be influenced by predicted climatic changes. We utilised Bayesian methodology and spatial genetic landscapes to establish broad-scale patterns and show that the western CFR is a contact zone for several clades in all three taxa, supporting the hypothesis of phylogeographic congruence. Current levels of gene flow are virtually zero between the western and eastern CFR. In the east, gene flow between populations is negligible at present but was probably stronger in the past given the present lack of strong genetic structure. Bioclimatic modelling predicted that climatically suitable areas within the CFR will decline for
Bradypodion spp. and
P. burchelli, with areas high in clade turnover loosing more climatically suitable areas than areas with low clade turnover. The models also predict that loss of climatic suitability may result in highly fragmented and patchy distributions, resulting in a greater loss of connectivity. In contrast,
A. atra does not show significant climatic suitability losses overall, although it may experience localised losses (and gains). This species is not predicted to loose suitability in areas of high clade turnover. Thus, the incorporation of genetic data into climatic models has extended our knowledge on the vulnerability of these species given the predicted threat of landscape change.
Uncombable hair syndrome (UHS), also known as “spun glass hair syndrome,” “pili trianguli et canaliculi,” or “cheveux incoiffables” is a rare anomaly of the hair shaft that occurs in children and ...improves with age. UHS is characterized by dry, frizzy, spangly, and often fair hair that is resistant to being combed flat. Until now, both simplex and familial UHS-affected case subjects with autosomal-dominant as well as -recessive inheritance have been reported. However, none of these case subjects were linked to a molecular genetic cause. Here, we report the identification of UHS-causative mutations located in the three genes PADI3 (peptidylarginine deiminase 3), TGM3 (transglutaminase 3), and TCHH (trichohyalin) in a total of 11 children. All of these individuals carry homozygous or compound heterozygous mutations in one of these three genes, indicating an autosomal-recessive inheritance pattern in the majority of UHS case subjects. The two enzymes PADI3 and TGM3, responsible for posttranslational protein modifications, and their target structural protein TCHH are all involved in hair shaft formation. Elucidation of the molecular outcomes of the disease-causing mutations by cell culture experiments and tridimensional protein models demonstrated clear differences in the structural organization and activity of mutant and wild-type proteins. Scanning electron microscopy observations revealed morphological alterations in hair coat of Padi3 knockout mice. All together, these findings elucidate the molecular genetic causes of UHS and shed light on its pathophysiology and hair physiology in general.
We present a new statistical method to identify genes in which one or more variants influence quantitative traits. We use the Genetic Analysis Workshop 17 (GAW17) data set of unrelated individuals as ...a test of the method on the raw GAW17 phenotypes and on residuals after fitting linear models to individual-based covariates. By performing appropriate randomization tests, we found many significant results for a proportion of the genes that contain variants that directly contribute to disease but that have an increased type I error for analyses of raw phenotypes. Power calculations show that our methods have the ability to reliably identify a subset of the loci contributing to disease. When we applied our method to derived phenotypes, we removed many false positives, giving appropriate type I error rates at little cost to power. The correlation between genome-wide heterozygosity and the value of the trait Q1 appears to drive much of the type I error in this data set.
Enrichment of DNA by hybridisation is an important tool which enables users to gather target-focused next-generation sequence data in an economical fashion. Current in-solution methods capture short ...fragments of around 200-300 nt, potentially missing key structural information such as recombination or translocations often found in viral or bacterial pathogens. The increasing use of long-read third-generation sequencers requires methods and protocols to be adapted for their specific requirements. Here, we present a variation of the traditional bait-capture approach which can selectively enrich large fragments of DNA or cDNA from specific bacterial and viral pathogens, for sequencing on long-read sequencers. We enriched cDNA from cultured influenza virus A, human cytomegalovirus (HCMV) and genomic DNA from two strains of
(
) from a background of cell line or spiked human DNA. We sequenced the enriched samples on the Oxford Nanopore MinION™ and the Illumina MiSeq platform and present an evaluation of the method, together with analysis of the sequence data. We found that unenriched influenza A and HCMV samples had no reads matching the target organism due to the high background of DNA from the cell line used to culture the pathogen. In contrast, enriched samples sequenced on the MinION™ platform had 57 % and 99 % best-quality on-target reads respectively.
The niche concept implies that a relationship exists between a species and its environment, while macro-ecological theory suggests that an important attribute of a species' environment is climate. ...Thus, changes in climate could affect individual species, but also communities. Here, we analysed the potential impacts of climate change on dwarf chameleons. A niche-based modelling technique was used to predict current suitable climatic habitat for most Bradypodion species and for their phylogenetic clades. Additionally, the models were projected into the future (2080) using the IPCC climate change scenarios. All models for Bradypodion species and clades showed responses to predicted climate change, however, the degree and extent of these responses were individualistic. Most species responded with a contraction in predicated climatic suitability, but some registered an expansion or a shift. These results have important implications in understanding the vulnerability of biodiversity to climate change, and for the importance of considering the effects of predicted climatic shifts on the protection of biodiversity.
Congenital cardiovascular malformation (CVM) affects 7/1000 live births. Approximately 20% of cases are caused by chromosomal and syndromic conditions. Rare Mendelian families segregating particular ...forms of CVM have also been described. Among the remaining 80% of non-syndromic cases, there is a familial predisposition implicating as yet unidentified genetic factors. Since the reproductive consequences to an individual of CVM are usually severe, evolutionary considerations suggest predisposing variants are likely to be rare. The overall aim of my PhD was to use next generation sequencing (NGS) methods to identify such rare, potentially disease causing variants in CVM. First, I developed a novel approach to calculate the sensitivity and specificity of NGS data in detecting variants using publicly available population frequency data. My aim was to provide a method that would yield sound estimates of the quality of a sequencing experiment without the need for additional genotyping in the sequenced samples. I developed such a method and demonstrated that it provided comparable results to methods using microarray data as a reference. Furthermore, I evaluated different variant calling pipelines and showed that they have a large effect on sensitivity and specificity. Following this, the NovoAlign-Samtools and BWA-Dindel pipelines were used to identify single base substitution and indel variants in three pedigrees, where predisposition to a different disease appears to segregate following an autosomal dominant mode of inheritance. I identified potentially causative variants segregating with disease in all three of the pedigrees. In the pedigrees with Dilated Cardiomyopathy and Hereditary Sclerosing Poikiloderma these variants were in plausible candidate genes. Finally, NGS was used to identify rare, potentially disease causing indel variants in patients with sporadic, non-syndromic forms of CVM characterised by chamber hypoplasia. Two indel calling pipelines were used as a means to increase confidence in the identified indels. These two pipelines achieved the highest sensitivity calls using the method described above. In the 133 cases, evaluated for 403 candidate genes, indels were identified in 4 known causative genes for human cardiovascular disease, namely MYL1, NOTCH1, TNNT2, and DSC2.
Congenital cardiovascular malformation (CVM) affects 7/1000 live births. Approximately 20% of cases are caused by chromosomal and syndromic conditions. Rare Mendelian families segregating particular ...forms of CVM have also been described. Among the remaining 80% of non-syndromic cases, there is a familial predisposition implicating as yet unidentified genetic factors. Since the reproductive consequences to an individual of CVM are usually severe, evolutionary considerations suggest predisposing variants are likely to be rare. The overall aim of my PhD was to use next generation sequencing (NGS) methods to identify such rare, potentially disease causing variants in CVM. First, I developed a novel approach to calculate the sensitivity and specificity of NGS data in detecting variants using publicly available population frequency data. My aim was to provide a method that would yield sound estimates of the quality of a sequencing experiment without the need for additional genotyping in the sequenced samples. I developed such a method and demonstrated that it provided comparable results to methods using microarray data as a reference. Furthermore, I evaluated different variant calling pipelines and showed that they have a large effect on sensitivity and specificity. Following this, the NovoAlign-Samtools and BWA-Dindel pipelines were used to identify single base substitution and indel variants in three pedigrees, where predisposition to a different disease appears to segregate following an autosomal dominant mode of inheritance. I identified potentially causative variants segregating with disease in all three of the pedigrees. In the pedigrees with Dilated Cardiomyopathy and Hereditary Sclerosing Poikiloderma these variants were in plausible candidate genes. Finally, NGS was used to identify rare, potentially disease causing indel variants in patients with sporadic, non-syndromic forms of CVM characterised by chamber hypoplasia. Two indel calling pipelines were used as a means to increase confidence in the identified indels. These two pipelines achieved the highest sensitivity calls using the method described above. In the 133 cases, evaluated for 403 candidate genes, indels were identified in 4 known causative genes for human cardiovascular disease, namely MYL1, NOTCH1, TNNT2, and DSC2.
The niche concept implies that a relationship exists between a species and its environment, while macro-ecological theory suggests that an important attribute of a species' environment is climate. ...Thus, changes in climate could affect individual species, but also communities. Here, we analysed the potential impacts of climate change on dwarf chameleons. A niche-based modelling technique was used to predict current suitable climatic habitat for most Bradypodion species and for their phylogenetic clades. Additionally, the models were projected into the future (2080) using the IPCC climate change scenarios. All models for Bradypodion species and clades showed responses to predicted climate change, however, the degree and extent of these responses were individualistic. Most species responded with a contraction in predicated climatic suitability, but some registered an expansion or a shift. These results have important implications in understanding the vulnerability of biodiversity to climate change, and for the importance of considering the effects of predicted climatic shifts on the protection of biodiversity.
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