Browsing by Author "Freed NE"

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    A simple screen to identify promoters conferring high levels of phenotypic noise.
    (PUBLIC LIBRARY SCIENCE, 2008-12) Freed NE; Silander OK; Stecher B; Böhm A; Hardt W-D; Ackermann M
    Genetically identical populations of unicellular organisms often show marked variation in some phenotypic traits. To investigate the molecular causes and possible biological functions of this phenotypic noise, it would be useful to have a method to identify genes whose expression varies stochastically on a certain time scale. Here, we developed such a method and used it for identifying genes with high levels of phenotypic noise in Salmonella enterica ssp. I serovar Typhimurium (S. Typhimurium). We created a genomic plasmid library fused to a green fluorescent protein (GFP) reporter and subjected replicate populations harboring this library to fluctuating selection for GFP expression using fluorescent-activated cell sorting (FACS). After seven rounds of fluctuating selection, the populations were strongly enriched for promoters that showed a high amount of noise in gene expression. Our results indicate that the activity of some promoters of S. Typhimurium varies on such a short time scale that these promoters can absorb rapid fluctuations in the direction of selection, as imposed during our experiment. The genomic fragments that conferred the highest levels of phenotypic variation were promoters controlling the synthesis of flagella, which are associated with virulence and host-pathogen interactions. This confirms earlier reports that phenotypic noise may play a role in pathogenesis and indicates that these promoters have among the highest levels of noise in the S. Typhimurium genome. This approach can be applied to many other bacterial and eukaryotic systems as a simple method for identifying genes with noisy expression.
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    Combining Tn-seq with comparative genomics identifies proteins uniquely essential in Shigella flexneri
    (3/09/2015) Freed NE; Bumann D; Silander O
    Protein functions that are essential for the growth of bacterial pathogens provide promising targets for antibacterial treatment. This is especially true if those functions are uniquely essential for the pathogen, as this might allow the development of targeted antibiotics, i.e. those that disrupt essential functions only for the pathogenic bacteria. Here we present the results of a Tn-seq experiment designed to detect essential protein coding genes in Shigella flexneri 2a 2457T on a genome-wide scale. Our results suggest that 471 protein-coding genes in this organism are critical for cellular growth in rich media. Comparing this set of essential genes (the essential gene complement) with their orthologues in the closely related organism Escherichia coli K12 BW25113 revealed a significant number of genes that are essential in Shigella but not in E. coli, suggesting that the functional correspondence of these proteins had changed. Notably, we also identified a set of functionally related genes that are essential in Shigella but which have no orthologues in E. coli. We found an extreme bias in proteins that have evolved to provide essential functions, with many proteins essential in Shigella but not E. coli, but with none (or very few) being essential in E. coli but not Shigella. We also identify a set- of genes involved in nucleotide biosynthesis that are essential in Shigella, but which lack orthologues in E. coli. Consequently, the data presented here suggest that the essential gene complement can quickly become organism specific, especially for pathogenic organisms whose genomes might have reduced robustness in their metabolic capacity (e.g. functional redundancy), or a reduced numbers of protein coding genes. These results thus open the possibility of developing antibiotic treatments that target differentially essential genes, which may exist even between very closely related strains of bacteria.
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    Long-read sequencing reveals atypical mitochondrial genome structure in a New Zealand marine isopod
    (The Royal Society, 2022-01-12) Pearman WS; Wells SJ; Dale J; Silander OK; Freed NE
    Most animal mitochondrial genomes are small, circular and structurally conserved. However, recent work indicates that diverse taxa possess unusual mitochondrial genomes. In Isopoda, species in multiple lineages have atypical and rearranged mitochondrial genomes. However, more species of this speciose taxon need to be evaluated to understand the evolutionary origins of atypical mitochondrial genomes in this group. In this study, we report the presence of an atypical mitochondrial structure in the New Zealand endemic marine isopod, Isocladus armatus. Data from long- and short-read DNA sequencing suggest that I. armatus has two mitochondrial chromosomes. The first chromosome consists of two mitochondrial genomes that have been inverted and fused together in a circular form, and the second chromosome consists of a single mitochondrial genome in a linearized form. This atypical mitochondrial structure has been detected in other isopod lineages, and our data from an additional divergent isopod lineage (Sphaeromatidae) lends support to the hypothesis that atypical structure evolved early in the evolution of Isopoda. Additionally, we find that an asymmetrical site previously observed across many species within Isopoda is absent in I. armatus, but confirm the presence of two asymmetrical sites recently reported in two other isopod species.
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    Rapid and inexpensive whole-genome sequencing of SARS-CoV-2 using 1200 bp tiled amplicons and Oxford Nanopore Rapid Barcoding
    (Oxford University Press, 2020-07-18) Freed NE; Vlková M; Faisal MB; Silander OK
    Rapid and cost-efficient whole-genome sequencing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes coronavirus disease 2019, is critical for understanding viral transmission dynamics. Here we show that using a new multiplexed set of primers in conjunction with the Oxford Nanopore Rapid Barcode library kit allows for faster, simpler, and less expensive SARS-CoV-2 genome sequencing. This primer set results in amplicons that exhibit lower levels of variation in coverage compared to other commonly used primer sets. Using five SARS-CoV-2 patient samples with Cq values between 20 and 31, we show that high-quality genomes can be generated with as few as 10 000 reads (∼5 Mbp of sequence data). We also show that mis-classification of barcodes, which may be more likely when using the Oxford Nanopore Rapid Barcode library prep, is unlikely to cause problems in variant calling. This method reduces the time from RNA to genome sequence by more than half compared to the more standard ligation-based Oxford Nanopore library preparation method at considerably lower costs.
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    Tracking the international spread of SARS-CoV-2 lineages B.1.1.7 and B.1.351/501Y-V2 with grinch
    (F1000 Research Limited, 2021-09-17) O'Toole Á; Hill V; Pybus OG; Watts A; Bogoch II; Khan K; Messina JP; COVID-19 Genomics UK (COG-UK) consortium; Network for Genomic Surveillance in South Africa (NGS-SA); Brazil-UK CADDE Genomic Network; Tegally H; Lessells RR; Giandhari J; Pillay S; Tumedi KA; Nyepetsi G; Kebabonye M; Matsheka M; Mine M; Tokajian S; Hassan H; Salloum T; Merhi G; Koweyes J; Geoghegan JL; de Ligt J; Ren X; Storey M; Freed NE; Pattabiraman C; Prasad P; Desai AS; Vasanthapuram R; Schulz TF; Steinbrück L; Stadler T; Swiss Viollier Sequencing Consortium; Parisi A; Bianco A; García de Viedma D; Buenestado-Serrano S; Borges V; Isidro J; Duarte S; Gomes JP; Zuckerman NS; Mandelboim M; Mor O; Seemann T; Arnott A; Draper J; Gall M; Rawlinson W; Deveson I; Schlebusch S; McMahon J; Leong L; Lim CK; Chironna M; Loconsole D; Bal A; Josset L; Holmes E; St George K; Lasek-Nesselquist E; Sikkema RS; Oude Munnink B; Koopmans M; Brytting M; Sudha Rani V; Pavani S; Smura T; Heim A; Kurkela S; Umair M; Salman M; Bartolini B; Rueca M; Drosten C; Wolff T; Silander O; Eggink D; Reusken C; Vennema H; Park A; Carrington C; Sahadeo N; Carr M; Gonzalez G; SEARCH Alliance San Diego; National Virus Reference Laboratory; SeqCOVID-Spain; Danish Covid-19 Genome Consortium (DCGC); Communicable Diseases Genomic Network (CDGN); Dutch National SARS-CoV-2 surveillance program; Division of Emerging Infectious Diseases (KDCA); de Oliveira T; Faria N; Rambaut A; Kraemer MUG
    Late in 2020, two genetically-distinct clusters of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with mutations of biological concern were reported, one in the United Kingdom and one in South Africa. Using a combination of data from routine surveillance, genomic sequencing and international travel we track the international dispersal of lineages B.1.1.7 and B.1.351 (variant 501Y-V2). We account for potential biases in genomic surveillance efforts by including passenger volumes from location of where the lineage was first reported, London and South Africa respectively. Using the software tool grinch (global report investigating novel coronavirus haplotypes), we track the international spread of lineages of concern with automated daily reports, Further, we have built a custom tracking website (cov-lineages.org/global_report.html) which hosts this daily report and will continue to include novel SARS-CoV-2 lineages of concern as they are detected.