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Auto Tag: Whole genome sequencing

July 7, 2019

Combining de novo and reference-guided assembly with scaffold_builder.

Genome sequencing has become routine, however genome assembly still remains a challenge despite the computational advances in the last decade. In particular, the abundance of repeat elements in genomes makes it difficult to assemble them into a single complete sequence. Identical repeats shorter than the average read length can generally be assembled without issue. However, longer repeats such as ribosomal RNA operons cannot be accurately assembled using existing tools. The application Scaffold_builder was designed to generate scaffolds – super contigs of sequences joined by N-bases – based on the similarity to a closely related reference sequence. This is independent of mate-pair information and can be used complementarily for genome assembly, e.g. when mate-pairs are not available or have already been exploited. Scaffold_builder was evaluated using simulated pyrosequencing reads of the bacterial genomes Escherichia coli 042, Lactobacillus salivarius UCC118 and Salmonella enterica subsp. enterica serovar Typhi str. P-stx-12. Moreover, we sequenced two genomes from Salmonella enterica serovar Typhimurium LT2 G455 and Salmonella enterica serovar Typhimurium SDT1291 and show that Scaffold_builder decreases the number of contig sequences by 53% while more than doubling their average length. Scaffold_builder is written in Python and is available at http://edwards.sdsu.edu/scaffold_builder. A web-based implementation is additionally provided to allow users to submit a reference genome and a set of contigs to be scaffolded.

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July 7, 2019

Assemblathon 2: evaluating de novo methods of genome assembly in three vertebrate species.

The process of generating raw genome sequence data continues to become cheaper, faster, and more accurate. However, assembly of such data into high-quality, finished genome sequences remains challenging. Many genome assembly tools are available, but they differ greatly in terms of their performance (speed, scalability, hardware requirements, acceptance of newer read technologies) and in their final output (composition of assembled sequence). More importantly, it remains largely unclear how to best assess the quality of assembled genome sequences. The Assemblathon competitions are intended to assess current state-of-the-art methods in genome assembly.In Assemblathon 2, we provided a variety of sequence data to be assembled for three vertebrate species (a bird, a fish, and snake). This resulted in a total of 43 submitted assemblies from 21 participating teams. We evaluated these assemblies using a combination of optical map data, Fosmid sequences, and several statistical methods. From over 100 different metrics, we chose ten key measures by which to assess the overall quality of the assemblies.Many current genome assemblers produced useful assemblies, containing a significant representation of their genes and overall genome structure. However, the high degree of variability between the entries suggests that there is still much room for improvement in the field of genome assembly and that approaches which work well in assembling the genome of one species may not necessarily work well for another.

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July 7, 2019

Complete genome sequence of the Mesoplasma florum W37 strain.

Mesoplasma florum is a small-genome fast-growing mollicute that is an attractive model for systems and synthetic genomics studies. We report the complete 825,824-bp genome sequence of a second representative of this species, M. florum strain W37, which contains 733 predicted open reading frames and 35 stable RNAs.

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July 7, 2019

Enhanced 5-methylcytosine detection in single-molecule, real-time sequencing via Tet1 oxidation.

DNA methylation serves as an important epigenetic mark in both eukaryotic and prokaryotic organisms. In eukaryotes, the most common epigenetic mark is 5-methylcytosine, whereas prokaryotes can have 6-methyladenine, 4-methylcytosine, or 5-methylcytosine. Single-molecule, real-time sequencing is capable of directly detecting all three types of modified bases. However, the kinetic signature of 5-methylcytosine is subtle, which presents a challenge for detection. We investigated whether conversion of 5-methylcytosine to 5-carboxylcytosine using the enzyme Tet1 would enhance the kinetic signature, thereby improving detection.We characterized the kinetic signatures of various cytosine modifications, demonstrating that 5-carboxylcytosine has a larger impact on the local polymerase rate than 5-methylcytosine. Using Tet1-mediated conversion, we show improved detection of 5-methylcytosine using in vitro methylated templates and apply the method to the characterization of 5-methylcytosine sites in the genomes of Escherichia coli MG1655 and Bacillus halodurans C-125.We have developed a method for the enhancement of directly detecting 5-methylcytosine during single-molecule, real-time sequencing. Using Tet1 to convert 5-methylcytosine to 5-carboxylcytosine improves the detection rate of this important epigenetic marker, thereby complementing the set of readily detectable microbial base modifications, and enhancing the ability to interrogate eukaryotic epigenetic markers.

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July 7, 2019

Complete genome sequence of a multidrug-resistant Salmonella enterica serovar Typhimurium var. 5- strain isolated from chicken breast.

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5- CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.

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July 7, 2019

Genome of an arbuscular mycorrhizal fungus provides insight into the oldest plant symbiosis.

The mutualistic symbiosis involving Glomeromycota, a distinctive phylum of early diverging Fungi, is widely hypothesized to have promoted the evolution of land plants during the middle Paleozoic. These arbuscular mycorrhizal fungi (AMF) perform vital functions in the phosphorus cycle that are fundamental to sustainable crop plant productivity. The unusual biological features of AMF have long fascinated evolutionary biologists. The coenocytic hyphae host a community of hundreds of nuclei and reproduce clonally through large multinucleated spores. It has been suggested that the AMF maintain a stable assemblage of several different genomes during the life cycle, but this genomic organization has been questioned. Here we introduce the 153-Mb haploid genome of Rhizophagus irregularis and its repertoire of 28,232 genes. The observed low level of genome polymorphism (0.43 SNP per kb) is not consistent with the occurrence of multiple, highly diverged genomes. The expansion of mating-related genes suggests the existence of cryptic sex-related processes. A comparison of gene categories confirms that R. irregularis is close to the Mucoromycotina. The AMF obligate biotrophy is not explained by genome erosion or any related loss of metabolic complexity in central metabolism, but is marked by a lack of genes encoding plant cell wall-degrading enzymes and of genes involved in toxin and thiamine synthesis. A battery of mycorrhiza-induced secreted proteins is expressed in symbiotic tissues. The present comprehensive repertoire of R. irregularis genes provides a basis for future research on symbiosis-related mechanisms in Glomeromycota.

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July 7, 2019

Complete genome sequence of Staphylococcus aureus Z172, a vancomycin-intermediate and daptomycin-nonsusceptible methicillin-resistant strain isolated in Taiwan.

We report the complete genome sequence of Z172, a representative strain of sequence type 239-staphylococcal cassette chromosome mec type III (ST239-SCCmec type III) hospital-associated methicillin-resistant Staphylococcus aureus in Taiwan. Strain Z172 also exhibits a vancomycin-intermediate and daptomycin-nonsusceptible phenotype.

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July 7, 2019

Complete genome sequence of Leifsonia xyli subsp. cynodontis strain DSM46306, a gram-positive bacterial pathogen of grasses.

We announce the complete genome sequence of Leifsonia xyli subsp. cynodontis, a vascular pathogen of Bermuda grass. The species also comprises Leifsonia xyli subsp. xyli, a sugarcane pathogen. Since these two subspecies have genome sequences available, a comparative analysis will contribute to our understanding of the differences in their biology and host specificity.

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July 7, 2019

Draft genome of Spiribacter salinus M19-40, an abundant gammaproteobacterium in aquatic hypersaline environments.

We have previously used a de novo metagenomic assembly approach to describe the presence of an abundant gammaproteobacterium comprising nearly 15% of the microbial community in an intermediate salinity solar saltern pond. We have obtained this microbe in pure culture and describe the genome sequencing of the halophilic photoheterotrophic microbe, Spiribacter salinus M19-40.

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July 7, 2019

Mutation in the C-di-AMP cyclase dacA affects fitness and resistance of methicillin resistant Staphylococcus aureus.

Faster growing and more virulent strains of methicillin resistant Staphylococcus aureus (MRSA) are increasingly displacing highly resistant MRSA. Elevated fitness in these MRSA is often accompanied by decreased and heterogeneous levels of methicillin resistance; however, the mechanisms for this phenomenon are not yet fully understood. Whole genome sequencing was used to investigate the genetic basis of this apparent correlation, in an isogenic MRSA strain pair that differed in methicillin resistance levels and fitness, with respect to growth rate. Sequencing revealed only one single nucleotide polymorphism (SNP) in the diadenylate cyclase gene dacA in the faster growing but less resistant strain. Diadenylate cyclases were recently discovered to synthesize the new second messenger cyclic diadenosine monophosphate (c-di-AMP). Introduction of this mutation into the highly resistant but slower growing strain reduced resistance and increased its growth rate, suggesting a direct connection between the dacA mutation and the phenotypic differences of these strains. Quantification of cellular c-di-AMP revealed that the dacA mutation decreased c-di-AMP levels resulting in reduced autolysis, increased salt tolerance and a reduction in the basal expression of the cell wall stress stimulon. These results indicate that c-di-AMP affects cell envelope-related signalling in S. aureus. The influence of c-di-AMP on growth rate and methicillin resistance in MRSA indicate that altering c-di-AMP levels could be a mechanism by which MRSA strains can increase their fitness levels by reducing their methicillin resistance levels.

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