[Show abstract][Hide abstract] ABSTRACT: With the widespread availability of high-throughput sequencing technologies, sequencing projects have become pervasive in the molecular life sciences. The huge bulk of data generated daily must be analyzed further by biologists with skills in bioinformatics and by "embedded bioinformaticians," i.e., bioinformaticians integrated in wet lab research groups. Thus, students interested in molecular life sciences must be trained in the main steps of genomics: sequencing, assembly, annotation and analysis. To reach that goal, a practical course has been set up for master students at the University of Lausanne: the "Sequence a genome" class. At the beginning of the academic year, a few bacterial species whose genome is unknown are provided to the students, who sequence and assemble the genome(s) and perform manual annotation. Here, we report the progress of the first class from September 2010 to June 2011 and the results obtained by seven master students who specifically assembled and annotated the genome of Estrella lausannensis, an obligate intracellular bacterium related to Chlamydia. The draft genome of Estrella is composed of 29 scaffolds encompassing 2,819,825 bp that encode for 2233 putative proteins. Estrella also possesses a 9136 bp plasmid that encodes for 14 genes, among which we found an integrase and a toxin/antitoxin module. Like all other members of the Chlamydiales order, Estrella possesses a highly conserved type III secretion system, considered as a key virulence factor. The annotation of the Estrella genome also allowed the characterization of the metabolic abilities of this strictly intracellular bacterium. Altogether, the students provided the scientific community with the Estrella genome sequence and a preliminary understanding of the biology of this recently-discovered bacterial genus, while learning to use cutting-edge technologies for sequencing and to perform bioinformatics analyses.
Full-text · Article · Feb 2015 · Frontiers in Microbiology
[Show abstract][Hide abstract] ABSTRACT: Pseudomonas knackmussii B13 was the first strain to be isolated in 1974 that could degrade chlorinated aromatic hydrocarbons. This discovery was the prologue for subsequent characterization of numerous bacterial metabolic pathways, for genetic and biochemical studies, and which spurred ideas for pollutant bioremediation. In this study we determined the complete genome sequence of B13 using next generation sequencing technologies and optical mapping. Genome annotation indicated that B13 has a variety of metabolic pathways for degrading monoaromatic hydrocarbons including chlorobenzoate, aminophenol, anthranilate, and hydroxyquinol, but not polyaromatic compounds. Comparative genome analysis revealed that B13 is closest to Pseudomonas denitrificans and Pseudomonas aeruginosa. The B13 genome contains at least 8 genomic islands (prophages and integrative conjugative elements - ICE), which were absent in closely related pseudomonads. We confirm that two ICE are identical copies of the 103-kb self-transmissible element ICEclc that carries the genes for chlorocatechol metabolism. Comparison of ICEclc showed it is composed of a variable and a "core" region, which is very conserved among proteobacterial genomes, suggesting a widely distributed family of so far uncharacterized ICE. Resequencing of two spontaneous B13 mutants revealed a number of single nucleotide substitutions, as well as excision of a large 220 kb region and a prophage, which drastically change the host metabolic capacity and survivability.
No preview · Article · May 2014 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Genotyping and molecular characterization of drug resistance mechanisms in Mycobacterium leprae enables disease transmission and drug resistance trends to be monitored. In the present study, we performed genome-wide analysis of Airaku-3, a multidrug resistant strain with an unknown mechanism of resistance to rifampicin. We identified 12 unique non-synonymous single-nucleotide polymorphisms (SNPs) including two in the transporter-encoding ctpC and ctpI genes. In addition, two SNPs were found that improve the resolution of SNP-based genotyping, particularly for Venezuelan and South-East Asian strains of M. leprae. This article is protected by copyright. All rights reserved.
Full-text · Article · Feb 2014 · Clinical Microbiology and Infection
[Show abstract][Hide abstract] ABSTRACT: To investigate the mechanism(s) of resistance to the RAF-inhibitor vemurafenib, we performed a comprehensive analysis of the genetic alterations occurring in metastatic lesions from a patient with a BRAFV600E-mutant cutaneous melanoma who, after a first response, underwent subsequent rechallenge with this drug.
We obtained blood and tissue samples from a patient diagnosed with a BRAFV600E-mutant cutaneous melanoma that was treated with vemurafenib and achieved a near-complete response. At progression, he received additional lines of chemo/immunotherapy and was successfully re-challenged with vemurafenib. Exome and RNA sequencing were performed on a pre-treatment tumor and two subcutaneous resistant metastases, one that was present at baseline and previously responded to vemurafenib (PV1), and one that appeared de novo after reintroduction of the drug (PV2). A culture established from PV1 was also analyzed.
We identified two NRAS activating somatic mutations, Q61R and Q61K, affecting two main subpopulations in the metastasis PV1, and a BRAF alternative splicing, involving exons 4-10, in the metastasis PV2. These alterations, known to confer resistance to RAF inhibitors, were tumor-specific, mutually exclusive, and were not detected in pre-treatment tumor samples. In addition, the oncogenic PIK3CAH1047R mutation was detected in a subpopulation of PV1, but this mutation did not appear to play a major role in vemurafenib resistance in this metastasis.
This work describes the co-existence within the same patient of different molecular mechanisms of resistance to vemurafenib affecting different metastatic sites. These findings have direct implications for the clinical management of BRAF-mutant melanoma.
Preview · Article · Aug 2013 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Kenny-Caffey syndrome (KCS) and the similar but more severe osteocraniostenosis (OCS) are genetic conditions characterized by impaired skeletal development with small and dense bones, short stature, and primary hypoparathyroidism with hypocalcemia. We studied five individuals with KCS and five with OCS and found that all of them had heterozygous mutations in FAM111A. One mutation was identified in four unrelated individuals with KCS, and another one was identified in two unrelated individuals with OCS; all occurred de novo. Thus, OCS and KCS are allelic disorders of different severity. FAM111A codes for a 611 amino acid protein with homology to trypsin-like peptidases. Although FAM111A has been found to bind to the large T-antigen of SV40 and restrict viral replication, its native function is unknown. Molecular modeling of FAM111A shows that residues affected by KCS and OCS mutations do not map close to the active site but are clustered on a segment of the protein and are at, or close to, its outer surface, suggesting that the pathogenesis involves the interaction with as yet unidentified partner proteins rather than impaired catalysis. FAM111A appears to be crucial to a pathway that governs parathyroid hormone production, calcium homeostasis, and skeletal development and growth.
Full-text · Article · May 2013 · The American Journal of Human Genetics
[Show abstract][Hide abstract] ABSTRACT: Sphingomonas wittichii RW1 is a dibenzofuran and dibenzodioxin-degrading bacterium with potentially interesting properties for bioaugmentation of contaminated sites. In order to understand the capacity of the microorganism to survive in the environment we used a genome-wide transposon scanning approach. RW1 transposon libraries were generated with around 22 000 independent insertions. Libraries were grown for an average of 50 generations (five successive passages in batch liquid medium) with salicylate as sole carbon and energy source in presence or absence of salt stress at -1.5 MPa. Alternatively, libraries were grown in sand with salicylate, at 50% water holding capacity, for 4 and 10 days (equivalent to 7 generations). Library DNA was recovered from the different growth conditions and scanned by ultrahigh throughput sequencing for the positions and numbers of inserted transposed kanamycin resistance gene. No transposon reads were recovered in 579 genes (10% of all annotated genes in the RW1 genome) in any of the libraries, suggesting those to be essential for survival under the used conditions. Libraries recovered from sand differed strongly from those incubated in liquid batch medium. In particular, important functions for survival of cells in sand at the short term concerned nutrient scavenging, energy metabolism and motility. In contrast to this, fatty acid metabolism and oxidative stress response were essential for longer term survival of cells in sand. Comparison to transcriptome data suggested important functions in sand for flagellar movement, pili synthesis, trehalose and polysaccharide synthesis and putative cell surface antigen proteins. Interestingly, a variety of genes were also identified, interruption of which cause significant increase in fitness during growth on salicylate. One of these was an Lrp family transcription regulator and mutants in this gene covered more than 90% of the total library after 50 generations of growth on salicylate. Our results demonstrate the power of genome-wide transposon scanning approaches for analysis of complex traits.
Full-text · Article · Mar 2013 · Environmental Microbiology
[Show abstract][Hide abstract] ABSTRACT: Animated profiles for the Bmal1, Reverbα (Nr1d1), and mPer1 genes for the Pol2 (red) occupancy and H3K4me3 (green) and H3K36me3 (blue) marks. The data were interpolated in time using spline interpolation (every 30 min).
[Show abstract][Hide abstract] ABSTRACT: Temporal relationship of Pol II promoter loadings and gene body density. (A) H3K36me3 signal in the most 3′-proximal 40% of gene bodies versus Pol II promoter occupancy at ZT2. There is a strong correlation between high Pol II loading at the promoter and high levels of H3K36me3 in the most 3′-proximal 40% of gene bodies. (B) mRNA expression versus Pol II promoter occupancy at ZT2 shows two distinct populations. (C) Pol II occupancies at promoters and in gene bodies vary in synchrony in a genome-wide fashion. Each gene is represented by a line indicating the orientation and the amplitude of changes during a diurnal cycle. Orientation is indicated by the color (key in upper right corner). (D–G) The genes in (A) were separated into groups according to orientation, from genes showing the largest variation in Pol II occupancy within gene bodies (lines with near vertical orientation in D) to genes showing the largest variation within promoter regions (lines with near horizontal orientation in F). Panel G shows genes for which gene body occupancy decreased as promoter region occupancy increased. Most highly expressed genes show patterns of concomitant changes in both promoter and body occupancy (panels D and E). (H) Idem as (C) but for the PAS1K signals against the promoter Pol II signals.
[Show abstract][Hide abstract] ABSTRACT: Comparison of class 1 and class 3 genes with the Hughes et al. 2009  and Hughes et al. 2012  gene sets. (A) Venn diagram showing the intersection between the genes of class 1 or 3—that is, transcripts that show diurnal variations of mRNA (Figure 6, n = 892, red) and the rhythmic transcripts in Hughes et al. 2009 (blue) and Hughes et al. 2012. In all pairwise comparisons, the percentage overlaps refer to the smallest of the two sets. Indicated p values for the overlaps are computed using the hypergeometric test. The gene sets and overlaps are given in Table S4. (B) The overlap is stratified according to decreasing mRNA amplitudes (peak to trough).
[Show abstract][Hide abstract] ABSTRACT: Pol II, H3K4me3, and H3K63me3 quantifications after normalization. The signals for each transcript per time point and per mark are given as described in Materials and Methods.
[Show abstract][Hide abstract] ABSTRACT: A model predicts temporal patterns for recruitment- or initiation-regulated rhythmic transcription. (A) Temporal variation of Pol II occupancy at promoter proximal positions (x,y,z) and gene bodies (w) as predicted by a simplified model of transcription. The model describes the (reversible) recruitment of polymerases to promoters, the (irreversible) transition to the open complex, the (irreversible) promoter escape, and the (irreversible) transition from a pausing to an elongating state, after which the polymerases travel to the end of the transcript. We investigate the two scenarios in which either the recruitment or the elongation rates are subject to cyclic circadian variation. (B) The mathematical model of transcription and its parameters: kf (forward recruitment rate), kb (backward recruitment rate), ko (isomerization rate), ki (promoter escape rate), kd (de-pausing rate), v (elongation rate). (C) Simulated temporal variation of occupancy in promoter proximal positions (x,y,z) and gene body (w) when the recruitment rate is varied in a circadian manner. (D) Idem when the de-pausing rate is varied in a circadian manner. The numerical values used in the simulation are kf = 1/min; kb = 0; ko = 0.1/s; ki = 1/min; kd = 1/min; v = 1/(6 s).
[Show abstract][Hide abstract] ABSTRACT: Temporal relationships of Pol II, H3K4me3, H3K36me3 profiles, and mRNA accumulation in mouse liver. Idem as Figure 5 with an extended selection of genes (n = 752, p<0.018, FDR = 0.5).
[Show abstract][Hide abstract] ABSTRACT: Data and gene lists for Figures 5 and 6. Note that the A0 values use background-subtracted signals (background values were 21 for mRNA expression and 0.47 for Pol II signals).