Nontuberculous mycobacteria (NTM) pose a threat to individuals with cystic fibrosis (CF) due to an increased prevalence of pulmonary infections, innate drug resistance of the bacteria, and potential transmission between CF patients. To explore the genetic diversity of NTM isolated from CF patients within the United States (US) and to identify potential transmission events, we sequenced and analyzed the genomes of 341 NTM isolates from 191 CF patients as part of a nationwide surveillance study. The most abundant species in the isolate cohort were Mycobacterium abscessus (59.5%), followed by species in the Mycobacterium avium complex (37.5%). Phylogenomic analyses of the three M. abscessus subspecies revealed that more than half of CF patients had isolates in one of four dominant clones, including two dominant clones of M. abscessus subspecies abscessus and two dominant clones of M. abscessus subspecies massiliense. M. avium isolates from US CF patients, however, do not have dominant clones and are phylogenetically diverse. Longitudinal NTM isolates were compared to determine genome-wide single nucleotide polymorphisms (SNPs) that occur within patients over time. This information was used to compare between and within-patient SNP distributions, to quantitatively define SNP thresholds suggestive of transmission, and calculate a posterior probability of recent transmission given the SNP distance between two isolates from different patients. Out of 114 patients with M. abscessus subspecies, ten clusters of highly similar isolates from 26 patients were identified. Among the 26 patients in the M. abscessus clusters, 12 attended the same CF care centers. No highly similar isolate clusters were observed in M. avium. Our study reveals the contrasting genomic diversity and epidemiology of two major NTM taxa and the potential for between-patient exposure and cross-transmission of these emerging pathogens.
Hibernation in sciurid rodents is a dynamic phenotype timed by a circannual clock. When housed in an animal facility, 13-lined ground squirrels exhibit variation in seasonal onset of hibernation, which is not explained by environmental or biological factors. We hypothesized that genetic factors instead drive variation in timing. After increasing genome contiguity, here, we employ a genotype-by-sequencing approach to characterize genetic variation in 153 ground squirrels. Combined with datalogger records (n = 72), we estimate high heritability (61–100%) for hibernation onset. Applying a genome-wide scan with 46,996 variants, we identify 2 loci significantly (p < 7.14 × 10−6), and 12 loci suggestively (p < 2.13 × 10−4), associated with onset. At the most significant locus, whole-genome resequencing reveals a putative causal variant in the promoter of FAM204A. Expression quantitative trait loci (eQTL) analyses further reveal gene associations for 8/14 loci. Our results highlight the power of applying genetic mapping to hibernation and present new insight into genetics driving its onset.
Background: Short-read whole genome sequencing (WGS) is a vital tool for clinical applications and basic research. Genetic divergence from the reference genome, repetitive sequences, and sequencing bias, reduce the performance of variant calling using short-read alignment, but the loss in recall and specificity has not been adequately characterized. For the clonal pathogen Mycobacterium tuberculosis (Mtb), researchers frequently exclude 10.7% of the genome believed to be repetitive and prone to erroneous variant calls. To benchmark short-read variant calling, we used 36 diverse clinical Mtb isolates dually sequenced with Illumina short-reads and PacBio long-reads. We systematically study the short-read variant calling accuracy and the influence of sequence uniqueness, reference bias, and GC content. Results: Reference based Illumina variant calling had a recall ≥89.0% and precision ≥98.5% across parameters evaluated. The best balance between precision and recall was achieved by tuning the mapping quality (MQ) threshold, i.e. confidence of the read mapping (recall 85.8%, precision 99.1% at MQ ≥ 40). Masking repetitive sequence content is an alternative conservative approach to variant calling that maintains high precision (recall 70.2%, precision 99.6% at MQ≥40). Of the genomic positions typically excluded for Mtb, 68% are accurately called using Illumina WGS including 52 of the 168 PE/PPE genes (34.5%). We present a refined list of low confidence regions and examine the largest sources of variant calling error. Conclusions: Our improved approach to variant calling has broad implications for the use of WGS in the study of Mtb biology, inference of transmission in public health surveillance systems, and more generally for WGS applications in other organisms.
Mycobacterium kubicae is 1 of nearly 200 species of nontuberculous mycobacteria (NTM), environmental micro-organisms that in some situations can infect humans and cause severe lung, skin and soft tissue infections. Although numerous studies have investigated the genetic variation among prevalent clinical NTM species, including Mycobacterium abscessus and Mycobacterium avium , many of the less common but clinically relevant NTM species, including M. kubicae , still lack complete genomes to serve as a comparative reference. Well-characterized representative genomes for each NTM species are important both for investigating the pathogenic potential of NTM, as well as for use in diagnostic methods, even for species that less frequently cause human disease. Here, we report the complete genomes of two M. kubicae strains, isolated from two unrelated patients. Hybrid short-read and long-read sequencing and assembly, using sequence reads from Illumina and Oxford Nanopore Technologies platforms, were utilized to resolve the chromosome and plasmid sequences of each isolate. The genome of NJH_MKUB1 had 5135 coding sequences (CDSs), a circular chromosome of length 5.3 Mb and two plasmids. The genome of NJH_MKUB2 had 5957 CDSs, a circular chromosome of 6.0 Mb and five plasmids. We compared our completed genomic assemblies to four recently released draft genomes of M. kubicae in order to better understand intraspecies genomic conservation and variability. We also identified genes implicated in drug resistance, virulence and persistence in the M. kubicae chromosome and plasmids. Virulence factors encoded in the genome and in the plasmids of M. kubicae provide a foundation for investigating how opportunistic environmental NTM may cause disease.
Although uncommon, nontuberculous mycobacterial (NTM) pulmonary infection in the Hawaiian Islands has a relatively high incidence and mortality compared to the mainland U.S. As a result, this study examines the possible geological and hydrological pathways by which NTM patients may become infected, including the environmental conditions that may favor growth and transport. Previously suggested infection routes include the inhalation of NTM attached to micro-droplets from infected home plumbing systems and aerosolized dust from garden soil. In this study we evaluate the possible routes NTM may take from riparian environments, into groundwater, into public water supplies and then into homes. Because NTM are notoriously hydrophobic and prone to attach to surfaces, mineralogy and surface chemistry of suspended sediment in streams, soils, and rock scrapings suggest that NTM may especially attach to Fe-oxides/hydroxides, and be transported as particles from losing streams to the aquifer on timescales of minutes to days. Within the aquifer, flow models indicate that water may be drawn into production wells on time scales (months) that permit NTM to survive and enter domestic water supplies. These processes depend on the presence of interconnected fracture networks with sufficient aperture to preclude complete autofiltration. The common occurrence of NTM in and around streams, in addition to wells, implies that the natural and built environments are capable of introducing a source of NTM into domestic water supplies via groundwater withdrawals. This may produce a persistent source of NTM infection to individuals through the presence of NTM-laden biofilms in home plumbing.
49Hibernation is a highly dynamic phenotype whose timing, for many mammals, is 50 controlled by a circannual clock and accompanied by rhythms in body mass and food intake. 51When housed in an animal facility, 13-lined ground squirrels exhibit individual variation in the 52 seasonal onset of hibernation, which is not explained by environmental or biological factors, 53 such as body mass and sex. We hypothesized that underlying genetic architecture instead 54 drives variation in this timing. After first increasing the contiguity of the genome assembly, we 55 therefore employed a genotype-by-sequencing approach to characterize genetic variation in 56 153 13-lined ground squirrels. Combining this with datalogger records, we estimated high 57 heritability (61-100%) for the seasonal onset of hibernation. After applying a genome-wide 58 scan with 46,996 variants, we also identified 21 loci significantly associated with hibernation 59 immergence, which alone accounted for 54% of the variance in the phenotype. The most 60 significant marker (SNP 15, p=3.81x10 −6 ) was located near prolactin-releasing hormone 61 receptor (PRLHR), a gene that regulates food intake and energy homeostasis. Other 62 significant loci were located near genes functionally related to hibernation physiology, including 63 muscarinic acetylcholine receptor M2 (CHRM2), involved in the control of heart rate, exocyst 64 complex component 4 (EXOC4) and prohormone convertase 2 (PCSK2), both of which are 65 involved in insulin signaling and processing. Finally, we applied an expression quantitative loci 66 (eQTL) analysis using existing transcriptome datasets, and we identified significant (q<0.1) 67 associations for 9/21 variants. Our results highlight the power of applying a genetic mapping 68 strategy to hibernation and present new insight into the genetics driving its seasonal onset. 69 70 Introduction 71
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