Rapid Whole-Genome Sequencing of Mycobacterium tuberculosis Isolates Directly from Clinical Samples.

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.

Using population data for assessing next-generation sequencing performance.

MOTIVATION: During the past 4 years, whole-exome sequencing has become a standard tool for finding rare variants causing Mendelian disorders. In that time, there has also been a proliferation of both sequencing platforms and approaches to analyse their output. This requires approaches to assess the performance of different methods. Traditionally, criteria such as comparison with microarray data or a number of known polymorphic sites have been used. Here we expand such approaches, developing a maximum likelihood framework and using it to estimate the sensitivity and specificity of whole-exome sequencing data. RESULTS: Using whole-exome sequencing data for a panel of 19 individuals, we show that estimated sensitivity and specificity are similar to those calculated using microarray data as a reference. We explore the effect of frequency misspecification arising from using an inappropriately selected population and find that, although the estimates are affected, the rankings across procedures remain the same. AVAILABILITY AND IMPLEMENTATION: An implementation using Perl and R can be found at busso.ncl.ac.uk (Username: igm101; Password: Z1z1nts).

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

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.

Finding genes that influence quantitative traits with tree-based clustering.

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.

The potential for predicted climate shifts to impact genetic landscapes of lizards in the South African Cape Floristic Region.

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.