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July 7, 2019

Real-time tRNA transit on single translating ribosomes at codon resolution.

Translation by the ribosome occurs by a complex mechanism involving the coordinated interaction of multiple nucleic acid and protein ligands. Here we use zero-mode waveguides (ZMWs) and sophisticated detection instrumentation to allow real-time observation of translation at physiologically relevant micromolar ligand concentrations. Translation at each codon is monitored by stable binding of transfer RNAs (tRNAs)-labelled with distinct fluorophores-to translating ribosomes, which allows direct detection of the identity of tRNA molecules bound to the ribosome and therefore the underlying messenger RNA (mRNA) sequence. We observe the transit of tRNAs on single translating ribosomes and determine the number of tRNA molecules simultaneously bound to the ribosome, at each codon of an mRNA molecule. Our results show that ribosomes are only briefly occupied by two tRNA molecules and that release of deacylated tRNA from the exit (E) site is uncoupled from binding of aminoacyl-tRNA site (A-site) tRNA and occurs rapidly after translocation. The methods outlined here have broad application to the study of mRNA sequences, and the mechanism and regulation of translation.

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July 7, 2019

Long, processive enzymatic DNA synthesis using 100% dye-labeled terminal phosphate-linked nucleotides.

We demonstrate the efficient synthesis of DNA with complete replacement of the four deoxyribonucleoside triphosphate (dNTP) substrates with nucleotides carrying fluorescent labels. A different, spectrally separable fluorescent dye suitable for single molecule fluorescence detection was conjugated to each of the four dNTPs via linkage to the terminal phosphate. Using these modified nucleotides, DNA synthesis by phi 29 DNA polymerase was observed to be processive for products thousands of bases in length, with labeled nucleotide affinities and DNA polymerization rates approaching unmodified dNTP levels. Results presented here show the compatibility of these nucleotides for single-molecule, real-time DNA sequencing applications.

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July 7, 2019

Accurate selfcorrection of errors in long reads using de Bruijn graphs.

New long read sequencing technologies, like PacBio SMRT and Oxford NanoPore, can produce sequencing reads up to 50,000 bp long but with an error rate of at least 15%. Reducing the error rate is necessary for subsequent utilisation of the reads in, e.g., de novo genome assembly. The error correction problem has been tackled either by aligning the long reads against each other or by a hybrid approach that uses the more accurate short reads produced by second generation sequencing technologies to correct the long reads.We present an error correction method that uses long reads only. The method consists of two phases: first we use an iterative alignment-free correction method based on de Bruijn graphs with increasing length of k-mers, and second, the corrected reads are further polished using long-distance dependencies that are found using multiple alignments. According to our experiments the proposed method is the most accurate one relying on long reads only for read sets with high coverage. Furthermore, when the coverage of the read set is at least 75x, the throughput of the new method is at least 20% higher.LoRMA is freely available at http://www.cs.helsinki.fi/u/lmsalmel/LoRMA/ CONTACT: leena.salmela@cs.helsinki.fi. © The Author(s) 2016. Published by Oxford University Press.

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July 7, 2019

Next-generation polyploid phylogenetics: rapid resolution of hybrid polyploid complexes using PacBio single-molecule sequencing.

Difficulties in generating nuclear data for polyploids have impeded phylogenetic study of these groups. We describe a high-throughput protocol and an associated bioinformatics pipeline (Pipeline for Untangling Reticulate Complexes (Purc)) that is able to generate these data quickly and conveniently, and demonstrate its efficacy on accessions from the fern family Cystopteridaceae. We conclude with a demonstration of the downstream utility of these data by inferring a multi-labeled species tree for a subset of our accessions. We amplified four c. 1-kb-long nuclear loci and sequenced them in a parallel-tagged amplicon sequencing approach using the PacBio platform. Purc infers the final sequences from the raw reads via an iterative approach that corrects PCR and sequencing errors and removes PCR-mediated recombinant sequences (chimeras). We generated data for all gene copies (homeologs, paralogs, and segregating alleles) present in each of three sets of 50 mostly polyploid accessions, for four loci, in three PacBio runs (one run per set). From the raw sequencing reads, Purc was able to accurately infer the underlying sequences. This approach makes it easy and economical to study the phylogenetics of polyploids, and, in conjunction with recent analytical advances, facilitates investigation of broad patterns of polyploid evolution.© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

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July 7, 2019

Enabling the democratization of the genomics revolution with a fully integrated web-based bioinformatics platform.

Continued advancements in sequencing technologies have fueled the development of new sequencing applications and promise to flood current databases with raw data. A number of factors prevent the seamless and easy use of these data, including the breadth of project goals, the wide array of tools that individually perform fractions of any given analysis, the large number of associated software/hardware dependencies, and the detailed expertise required to perform these analyses. To address these issues, we have developed an intuitive web-based environment with a wide assortment of integrated and cutting-edge bioinformatics tools in pre-configured workflows. These workflows, coupled with the ease of use of the environment, provide even novice next-generation sequencing users with the ability to perform many complex analyses with only a few mouse clicks and, within the context of the same environment, to visualize and further interrogate their results. This bioinformatics platform is an initial attempt at Empowering the Development of Genomics Expertise (EDGE) in a wide range of applications for microbial research.© The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

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July 7, 2019

Quantum changes in Helicobacter pylori gene expression accompany host-adaptation.

Helicobacter pylori is a highly successful gastric pathogen. High genomic plasticity allows its adaptation to changing host environments. Complete genomes of H. pylori clinical isolate UM032 and its mice-adapted serial derivatives 298 and 299, generated using both PacBio RS and Illumina MiSeq sequencing technologies, were compared to identify novel elements responsible for host-adaptation. The acquisition of a jhp0562-like allele, which encodes for a galactosyltransferase, was identified in the mice-adapted strains. Our analysis implies a new ß-1,4-galactosyltransferase role for this enzyme, essential for Ley antigen expression. Intragenomic recombination between babA and babB genes was also observed. Further, we expanded on the list of candidate genes whose expression patterns have been mediated by upstream homopolymer-length alterations to facilitate host adaption. Importantly, greater than four-fold reduction of mRNA levels was demonstrated in five genes. Among the down-regulated genes, three encode for outer membrane proteins, including BabA, BabB and HopD. As expected, a substantial reduction in BabA protein abundance was detected in mice-adapted strains 298 and 299 via Western analysis. Our results suggest that the expression of Ley antigen and reduced outer membrane protein expressions may facilitate H. pylori colonisation of mouse gastric epithelium.© The Author 2016. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

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July 7, 2019

Implementation and data analysis of Tn-seq, whole genome resequencing, and single-molecule real time sequencing for bacterial genetics.

Few discoveries have been more transformative to the biological sciences than the development of DNA sequencing technologies. The rapid advancement of sequencing and bioinformatics tools has revolutionized bacterial genetics, deepening our understanding of model and clinically relevant organisms. Although application of newer sequencing technologies to studies in bacterial genetics is increasing, the implementation of DNA sequencing technologies and development of the bioinformatics tools required for analyzing the large data sets generated remains a challenge for many. In this minireview, we have chosen to summarize three sequencing approaches that are particularly useful for bacterial genetics. We provide resources for scientists new to and interested in their application. Herein, we discuss the analysis of Tn-seq data to determine gene disruptions differentially represented in a mutant population, Illumina sequencing for identification of suppressor or other mutations, and we summarize single-molecule real time (SMRT) sequencing for de novo genome assembly and the use of the output data for detection of DNA base modifications. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

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July 7, 2019

Development of new methods for the quantitative detection and typing of Lactobacillus parabuchneri in dairy products

Thirty-one isolates of Lactobacillus parabuchneri were obtained from cheese containing histamine; of these, 26 were found to possess the hdcA gene encoding histidine decarboxylase. By analysing the genome data of 13 isolates, specific targets for the development of PCR-based detection and typing systems for L. parabuchneri were identified. The real-time PCR for detection showed a linear quantification over a range of 7 logs and a detection limit of 10 gene equivalents per reaction. The strain typing method utilised the amplification of repeat sequences and showed discrimination comparable with a phylogenetic tree, based on genome comparisons. The method was suitable for detecting and monitoring the development of L. parabuchneri in raw milk and cheese.

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July 7, 2019

Genomic analysis of the multi-drug-resistant clinical isolate Myroides odoratimimus PR63039.

Myroides odoratimimus (M. odoratimimus) has been gradually implicated as an important nosocomial pathogen that poses a serious health threat to immunocompromised patients owing to its multi-drug resistance. However, the resistance mechanism is currently unclear. To clarify the antibiotic resistance and infectivity mechanisms of M. odoratimimus, whole genome sequencing was performed on the multi-drug-resistant M. odoratimimus strain PR63039. The genome sequence was completed with single molecule real-time (SMRT) technologies. Then, annotation was performed using RAST and IMG-ER. A number of databases and software programs were used to analyze the genomic characteristics, including GC-Profile, ISfinder, CG viewer, ARDB, CARD, ResFinder, the VFDB database, PHAST and Progressive Mauve. The M. odoratimimus PR63039 genome consisted of a chromosome and a plasmid. The genome contained a large number of resistance genes and virulence factors. The distribution of the resistance genes was distinctive, and a resistance region named MY63039-RR was found. The subsystem features generated by RAST indicated that the annotated genome had 108 genes that were potentially involved in virulence, disease and defense, all of which had strong associations with resistance and pathogenicity. The prophage analysis showed two incomplete prophages in the genome. The genomic analysis of M. odoratimimus PR63039 partially clarified its antibiotic resistance mechanisms and virulence factors. Obtaining a clear understanding of its genomic characteristics will be conducive to the management of multidrug-resistant M. odoratimimus.

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July 7, 2019

What distinguishes cyanobacteria able to revive after desiccation from those that cannot: the genome aspect.

Filamentous cyanobacteria are the main founders and primary producers in biological desert soil crusts (BSCs) and are likely equipped to cope with one of the harshest environmental conditions on earth including daily hydration/dehydration cycles, high irradiance and extreme temperatures. Here, we resolved and report on the genome sequence of Leptolyngbya ohadii, an important constituent of the BSC. Comparative genomics identified a set of genes present in desiccation-tolerant but not in dehydration-sensitive cyanobacteria. RT qPCR analyses showed that the transcript abundance of many of them is upregulated during desiccation in L. ohadii. In addition, we identified genes where the orthologs detected in desiccation-tolerant cyanobacteria differs substantially from that found in desiccation-sensitive cells. We present two examples, treS and fbpA (encoding trehalose synthase and fructose 1,6-bisphosphate aldolase respectively) where, in addition to the orthologs present in the desiccation-sensitive strains, the resistant cyanobacteria also possess genes with different predicted structures. We show that in both cases the two orthologs are transcribed during controlled dehydration of L. ohadii and discuss the genetic basis for the acclimation of cyanobacteria to the desiccation conditions in desert BSC.© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

The genetic basis of anoxygenic photosynthetic arsenite oxidation.

‘Photoarsenotrophy’, the use of arsenite as an electron donor for anoxygenic photosynthesis, is thought to be an ancient form of phototrophy along with the photosynthetic oxidation of Fe(II), H2 S, H2 and NO2-. Photoarsenotrophy was recently identified from Paoha Island’s (Mono Lake, CA) arsenic-rich hot springs. The genomes of several photoarsenotrophs revealed a gene cluster, arxB2AB1CD, where arxA is predicted to encode for the sole arsenite oxidase. The role of arxA in photosynthetic arsenite oxidation was confirmed by disrupting the gene in a representative photoarsenotrophic bacterium, resulting in the loss of light-dependent arsenite oxidation. In situ evidence of active photoarsenotrophic microbes was supported by arxA mRNA detection for the first time, in red-pigmented microbial mats within the hot springs of Paoha Island. This work expands on the genetics for photosynthesis coupled to new electron donors and elaborates on known mechanisms for arsenic metabolism, thereby highlighting the complexities of arsenic biogeochemical cycling.© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.

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July 7, 2019

The comparative landscape of duplications in Heliconius melpomene and Heliconius cydno.

Gene duplications can facilitate adaptation and may lead to interpopulation divergence, causing reproductive isolation. We used whole-genome resequencing data from 34 butterflies to detect duplications in two Heliconius species, Heliconius cydno and Heliconius melpomene. Taking advantage of three distinctive signals of duplication in short-read sequencing data, we identified 744 duplicated loci in H. cydno and H. melpomene and evaluated the accuracy of our approach using single-molecule sequencing. We have found that duplications overlap genes significantly less than expected at random in H. melpomene, consistent with the action of background selection against duplicates in functional regions of the genome. Duplicate loci that are highly differentiated between H. melpomene and H. cydno map to four different chromosomes. Four duplications were identified with a strong signal of divergent selection, including an odorant binding protein and another in close proximity with a known wing colour pattern locus that differs between the two species. Heredity advance online publication, 7 December 2016; doi:10.1038/hdy.2016.107.

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July 7, 2019

Draft genome sequence of Mentha longifolia (L.) and development of resources for mint cultivar improvement.

The genus Mentha encompasses mint species cultivated for their essential oils, which are formulated into a vast array of consumer products. Desirable oil characteristics and resistance to the fungal disease Verticillium wilt are top priorities for the mint industry. However, cultivated mints have complex polyploid genomes and are sterile. Breeding efforts, therefore, require the development of genomic resources for fertile mint species. Here, we present draft de novo genome and plastome assemblies for a wilt-resistant South African accession of Mentha longifolia (L.) Huds., a diploid species ancestral to cultivated peppermint and spearmint. The 353 Mb genome contains 35 597 predicted protein-coding genes, including 292 disease resistance gene homologs, and nine genes determining essential oil characteristics. A genetic linkage map ordered 1397 genome scaffolds on 12 pseudochromosomes. More than two million simple sequence repeats were identified, which will facilitate molecular marker development. The M. longifolia genome is a valuable resource for both metabolic engineering and molecular breeding. This is exemplified by employing the genome sequence to clone and functionally characterize the promoters in a peppermint cultivar, and demonstrating the utility of a glandular trichome-specific promoter to increase expression of a biosynthetic gene, thereby modulating essential oil composition. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

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July 7, 2019

Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium.

From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned the multi locus sequence type ST796) was simultaneously isolated from geographically separate hospitals in south eastern Australia and New Zealand. Here we describe the complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate. We used PacBio single molecule real-time sequencing to establish a high quality, fully assembled genome comprising a circular chromosome of 2,888,087 bp and five plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233 is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-like ancestral progenitor by the accumulation or modification of five mosaic plasmids and five putative prophage, acquisition of two cryptic genomic islands, accrued chromosomal single nucleotide polymorphisms and a 80 kb region of recombination, also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and tetracycline respectively. The genomic dissection of this new clone presented here underscores the propensity of the hospital E. faecium lineage to change, presumably in response to the specific conditions of hospital and healthcare environments.

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July 7, 2019

Complete genome sequence of Lutibacter profundi LP1T isolated from an Arctic deep-sea hydrothermal vent system

Lutibacter profundi LP1T within the family Flavobacteriaceae was isolated from a biofilm growing on the surface of a black smoker chimney at the Loki’s Castle vent field, located on the Arctic Mid-Ocean Ridge. The complete genome of L. profundi LP1T is the first genome to be published within the genus Lutibacter. L. profundi LP1T consists of a single 2,966,978 bp circular chromosome with a GC content of 29.8%. The genome comprises 2,537 protein-coding genes, 40 tRNA species and 2 rRNA operons. The microaerophilic, organotrophic isolate contains genes for all central carbohydrate metabolic pathways. However, genes for the oxidative branch of the pentose-phosphate-pathway, the glyoxylate shunt of the tricarboxylic acid cycle and the ATP citrate lyase for reverse TCA are not present. L. profundi LP1T utilizes starch, sucrose and diverse proteinous carbon sources. In accordance, the genome harbours 130 proteases and 104 carbohydrate-active enzymes, indicating a specialization in degrading organic matter. Among a small arsenal of 24 glycosyl hydrolases, which offer the possibility to hydrolyse diverse poly- and oligosaccharides, a starch utilization cluster was identified. Furthermore, a variety of enzymes may be secreted via T9SS and contribute to the hydrolytic variety of the microorganism. Genes for gliding motility are present, which may enable the bacteria to move within the biofilm. A substantial number of genes encoding for extracellular polysaccharide synthesis pathways, curli fibres and attachment to surfaces could mediate adhesion in the biofilm and may contribute to the biofilm formation. In addition to aerobic respiration, the complete denitrification pathway and genes for sulphide oxidation e.g. sulphide:quinone reductase are present in the genome. sulphide:quinone reductase and denitrification may serve as detoxification systems allowing L. profundi LP1T to thrive in a sulphide and nitrate enriched environment. The information gained from the genome gives a greater insight in the functional role of L. profundi LP1T in the biofilm and its adaption strategy in an extreme environment.

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