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July 7, 2019  |  

Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation.

We have developed a sequencing method on the Pacific Biosciences RS sequencer (the PacBio) for small DNA molecules that avoids the need for a standard library preparation. To date this approach has been applied toward sequencing single-stranded and double-stranded viral genomes, bacterial plasmids, plasmid vector models for DNA-modification analysis, and linear DNA fragments covering an entire bacterial genome. Using direct sequencing it is possible to generate sequence data from as little as 1 ng of DNA, offering a significant advantage over current protocols which typically require 400-500 ng of sheared DNA for the library preparation.

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July 7, 2019  |  

Sensitive and specific single-molecule sequencing of 5-hydroxymethylcytosine.

We describe strand-specific, base-resolution detection of 5-hydroxymethylcytosine (5-hmC) in genomic DNA with single-molecule sensitivity, combining a bioorthogonal, selective chemical labeling method of 5-hmC with single-molecule, real-time (SMRT) DNA sequencing. The chemical labeling not only allows affinity enrichment of 5-hmC-containing DNA fragments but also enhances the kinetic signal of 5-hmC during SMRT sequencing. We applied the approach to sequence 5-hmC in a genomic DNA sample with high confidence.

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July 7, 2019  |  

Direct detection and sequencing of damaged DNA bases.

Products of various forms of DNA damage have been implicated in a variety of important biological processes, such as aging, neurodegenerative diseases, and cancer. Therefore, there exists great interest to develop methods for interrogating damaged DNA in the context of sequencing. Here, we demonstrate that single-molecule, real-time (SMRT®) DNA sequencing can directly detect damaged DNA bases in the DNA template – as a by-product of the sequencing method – through an analysis of the DNA polymerase kinetics that are altered by the presence of a modified base. We demonstrate the sequencing of several DNA templates containing products of DNA damage, including 8-oxoguanine, 8-oxoadenine, O6-methylguanine, 1-methyladenine, O4-methylthymine, 5-hydroxycytosine, 5-hydroxyuracil, 5-hydroxymethyluracil, or thymine dimers, and show that these base modifications can be readily detected with single-modification resolution and DNA strand specificity. We characterize the distinct kinetic signatures generated by these DNA base modifications.

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July 7, 2019  |  

Computational solutions to large-scale data management and analysis.

Today we can generate hundreds of gigabases of DNA and RNA sequencing data in a week for less than US$5,000. The astonishing rate of data generation by these low-cost, high-throughput technologies in genomics is being matched by that of other technologies, such as real-time imaging and mass spectrometry-based flow cytometry. Success in the life sciences will depend on our ability to properly interpret the large-scale, high-dimensional data sets that are generated by these technologies, which in turn requires us to adopt advances in informatics. Here we discuss how we can master the different types of computational environments that exist – such as cloud and heterogeneous computing – to successfully tackle our big data problems.

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July 7, 2019  |  

Complete genome analysis of Serratia marcescens RSC-14: A plant growth-promoting bacterium that alleviates cadmium stress in host plants.

Serratia marcescens RSC-14 is a Gram-negative bacterium that was previously isolated from the surface-sterilized roots of the Cd-hyperaccumulator Solanum nigrum. The strain stimulates plant growth and alleviates Cd stress in host plants. To investigate the genetic basis for these traits, the complete genome of RSC-14 was obtained by single-molecule real-time sequencing. The genome of S. marcescens RSC-14 comprised a 5.12-Mbp-long circular chromosome containing 4,593 predicted protein-coding genes, 22 rRNA genes, 88 tRNA genes, and 41 pseudogenes. It contained genes with potential functions in plant growth promotion, including genes involved in indole-3-acetic acid (IAA) biosynthesis, acetoin synthesis, and phosphate solubilization. Moreover, annotation using NCBI and Rapid Annotation using Subsystem Technology identified several genes that encode antioxidant enzymes as well as genes involved in antioxidant production, supporting the observed resistance towards heavy metals, such as Cd. The presence of IAA pathway-related genes and oxidative stress-responsive enzyme genes may explain the plant growth-promoting potential and Cd tolerance, respectively. This is the first report of a complete genome sequence of Cd-tolerant S. marcescens and its plant growth promotion pathway. The whole-genome analysis of this strain clarified the genetic basis underlying its phenotypic and biochemical characteristics, underpinning the beneficial interactions between RSC-14 and plants.

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July 7, 2019  |  

Analysis of the complete genome sequence of Nocardia seriolae UTF1, the causative agent of fish nocardiosis: The first reference genome sequence of the fish pathogenic Nocardia species.

Nocardiosis caused by Nocardia seriolae is one of the major threats in the aquaculture of Seriola species (yellowtail; S. quinqueradiata, amberjack; S. dumerili and kingfish; S. lalandi) in Japan. Here, we report the complete nucleotide genome sequence of N. seriolae UTF1, isolated from a cultured yellowtail. The genome is a circular chromosome of 8,121,733 bp with a G+C content of 68.1% that encodes 7,697 predicted proteins. In the N. seriolae UTF1 predicted genes, we found orthologs of virulence factors of pathogenic mycobacteria and human clinical Nocardia isolates involved in host cell invasion, modulation of phagocyte function and survival inside the macrophages. The virulence factor candidates provide an essential basis for understanding their pathogenic mechanisms at the molecular level by the fish nocardiosis research community in future studies. We also found many potential antibiotic resistance genes on the N. seriolae UTF1 chromosome. Comparative analysis with the four existing complete genomes, N. farcinica IFM 10152, N. brasiliensis HUJEG-1 and N. cyriacigeorgica GUH-2 and N. nova SH22a, revealed that 2,745 orthologous genes were present in all five Nocardia genomes (core genes) and 1,982 genes were unique to N. seriolae UTF1. In particular, the N. seriolae UTF1 genome contains a greater number of mobile elements and genes of unknown function that comprise the differences in structure and gene content from the other Nocardia genomes. In addition, a lot of the N. seriolae UTF1-specific genes were assigned to the ABC transport system. Because of limited resources in ocean environments, these N. seriolae UTF1 specific ABC transporters might facilitate adaptation strategies essential for marine environment survival. Thus, the availability of the complete N. seriolae UTF1 genome sequence will provide a valuable resource for comparative genomic studies of N. seriolae isolates, as well as provide new insights into the ecological and functional diversity of the genus Nocardia.

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July 7, 2019  |  

A spontaneous mutation in kdsD, a biosynthesis gene for 3 Deoxy-D-manno-Octulosonic Acid, occurred in a ciprofloxacin resistant strain of Francisella tularensis and caused a high level of attenuation in murine models of tularemia.

Francisella tularensis, a gram-negative facultative intracellular bacterial pathogen, is the causative agent of tularemia and able to infect many mammalian species, including humans. Because of its ability to cause a lethal infection, low infectious dose, and aerosolizable nature, F. tularensis subspecies tularensis is considered a potential biowarfare agent. Due to its in vitro efficacy, ciprofloxacin is one of the antibiotics recommended for post-exposure prophylaxis of tularemia. In order to identify therapeutics that will be efficacious against infections caused by drug resistant select-agents and to better understand the threat, we sought to characterize an existing ciprofloxacin resistant (CipR) mutant in the Schu S4 strain of F. tularensis by determining its phenotypic characteristics and sequencing the chromosome to identify additional genetic alterations that may have occurred during the selection process. In addition to the previously described genetic alterations, the sequence of the CipR mutant strain revealed several additional mutations. Of particular interest was a frameshift mutation within kdsD which encodes for an enzyme necessary for the production of 3-Deoxy-D-manno-Octulosonic Acid (KDO), an integral component of the lipopolysaccharide (LPS). A kdsD mutant was constructed in the Schu S4 strain. Although it was not resistant to ciprofloxacin, the kdsD mutant shared many phenotypic characteristics with the CipR mutant, including growth defects under different conditions, sensitivity to hydrophobic agents, altered LPS profiles, and attenuation in multiple models of murine tularemia. This study demonstrates that the KdsD enzyme is essential for Francisella virulence and may be an attractive therapeutic target for developing novel medical countermeasures.

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July 7, 2019  |  

Structural variation offers new home for disease associations and gene discovery

Following completion of the Human Genome Project, most studies of human genetic variation have centered on single nucleotide polymorphisms (SNPs). SNPs are numerous in individual genomes and serve as useful genetic markers in association studies across a population. These markers have been leveraged to identify genetic loci for disease risk and draw associations with numerous traits of interest. Despite their usefulness, SNPs do not tell the whole story. For example, most SNPs are associated with only a small increased risk of disease, and they usually cannot identify on their own which genes are causal. This has resulted in what many researchers have referred to as missing or hidden heritability.

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July 7, 2019  |  

Multiple and diverse vsp and vlp sequences in Borrelia miyamotoi, a hard tick-borne zoonotic pathogen.

Based on chromosome sequences, the human pathogen Borrelia miyamotoi phylogenetically clusters with species that cause relapsing fever. But atypically for relapsing fever agents, B. miyamotoi is transmitted not by soft ticks but by hard ticks, which also are vectors of Lyme disease Borrelia species. To further assess the relationships of B. miyamotoi to species that cause relapsing fever, I investigated extrachromosomal sequences of a North American strain with specific attention on plasmid-borne vsp and vlp genes, which are the underpinnings of antigenic variation during relapsing fever. For a hybrid approach to achieve assemblies that spanned more than one of the paralogous vsp and vlp genes, a database of short-reads from next-generation sequencing was supplemented with long-reads obtained with real-time DNA sequencing from single polymerase molecules. This yielded three contigs of 31, 16, and 11 kb, which each contained multiple and diverse sequences that were homologous to vsp and vlp genes of the relapsing fever agent B. hermsii. Two plasmid fragments had coding sequences for plasmid partition proteins that differed from each other from paralogous proteins for the megaplasmid and a small plasmid of B. miyamotoi. One of 4 vsp genes, vsp1, was present at two loci, one of which was downstream of a candiate prokaryotic promoter. A limited RNA-seq analysis of a population growing in the blood of mice indicated that of the 4 different vsp genes vsp1 was the one that was expressed. The findings indicate that B. miyamotoi has at least four types of plasmids, two or more of which bear vsp and vlp gene sequences that are as numerous and diverse as those of relapsing fever Borrelia. The database and insights from these findings provide a foundation for further investigations of the immune responses to this pathogen and of the capability of B. miyamotoi for antigenic variation.

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July 7, 2019  |  

Genome of Cnaphalocrocis medinalis granulovirus, the first Crambidae-infecting betabaculovirus isolated from rice leaffolder to sequenced.

Cnaphalocrocis medinalis is a major pest of rice in South and South-East Asia. Insecticides are the major means farmers use for management. A naturally occurring baculovirus, C. medinalis granulovirus (CnmeGV), has been isolated from the larvae and this has the potential for use as microbial agent. Here, we described the complete genome sequence of CnmeGV and compared it to other baculovirus genomes. The genome of CnmeGV is 112,060 base pairs in length, has a G+C content of 35.2%. It contains 133 putative open reading frames (ORFs) of at least 150 nucleotides. A hundred and one (101) of these ORFs are homologous to other baculovirus genes including 37 baculovirus core genes. Thirty-two (32) ORFs are unique to CnmeGV with no homologues detected in the GeneBank and 53 tandem repeats (TRs) with sequence length from 25 to 551 nt intersperse throughout the genome of CnmeGV. Six (6) homologous regions (hrs) were identified interspersed throughout the genome. Hr2 contains 11 imperfect palindromes and a high content of AT sequence (about 73%). The unique ORF28 contains a coiled-coil region and a zinc finger-like domain of 4-50 residues specialized by two C2C2 zinc finger motifs that putatively bound two atoms of zinc. ORF21 encoding a chit-1 protein suggesting a horizontal gene transfer from alphabaculovirus. The putative protein presents two carbohydrate-binding module family 14 (CBM_14) domains rather than other homologues detected from betabaculovirus that only contains one chit-binding region. Gene synteny maps showed the colinearity of sequenced betabaculovirus. Phylogenetic analysis indicated that CnmeGV grouped in the betabaculovirus, with a close relation to AdorGV. The cladogram obtained in this work grouped the 17 complete GV genomes in one monophyletic clade. CnmeGV represents a new crambidae host-isolated virus species from the genus Betabaculovirus and is most closely relative of AdorGV. The analyses and information derived from this study will provide a better understanding of the pathological symptoms caused by this virus and its potential use as a microbial pesticide.

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July 7, 2019  |  

Complete genome sequence and comparative genomics of a novel Myxobacterium Myxococcus hansupus.

Myxobacteria, a group of Gram-negative aerobes, belong to the class d-proteobacteria and order Myxococcales. Unlike anaerobic d-proteobacteria, they exhibit several unusual physiogenomic properties like gliding motility, desiccation-resistant myxospores and large genomes with high coding density. Here we report a 9.5 Mbp complete genome of Myxococcus hansupus that encodes 7,753 proteins. Phylogenomic and genome-genome distance based analysis suggest that Myxococcus hansupus is a novel member of the genus Myxococcus. Comparative genome analysis with other members of the genus Myxococcus was performed to explore their genome diversity. The variation in number of unique proteins observed across different species is suggestive of diversity at the genus level while the overrepresentation of several Pfam families indicates the extent and mode of genome expansion as compared to non-Myxococcales d-proteobacteria.

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July 7, 2019  |  

Population structure and antimicrobial resistance profiles of Streptococcus suis serotype 2 sequence type 25 strains

Strains of serotype 2 Streptococcus suis are responsible for swine and human infections. Different serotype 2 genetic backgrounds have been defined using multilocus sequence typing (MLST). However, little is known about the genetic diversity within each MLST sequence type (ST). Here, we used whole-genome sequencing to test the hypothesis that S. suis serotype 2 strains of the ST25 lineage are genetically heterogeneous. We evaluated 51 serotype 2 ST25 S. suis strains isolated from diseased pigs and humans in Canada, the United States of America, and Thailand. Whole-genome sequencing revealed numerous large-scale rearrangements in the ST25 genome, compared to the genomes of ST1 and ST28 S. suis strains, which result, among other changes, in disruption of a pilus island locus. We report that recombination and lateral gene transfer contribute to ST25 genetic diversity. Phylogenetic analysis identified two main and distinct Thai and North American clades grouping most strains investigated. These clades also possessed distinct patterns of antimicrobial resistance genes, which correlated with acquisition of different integrative and conjugative elements (ICEs). Some of these ICEs were found to be integrated at a recombination hot spot, previously identified as the site of integration of the 89K pathogenicity island in serotype 2 ST7 S. suis strains. Our results highlight the limitations of MLST for phylogenetic analysis of S. suis, and the importance of lateral gene transfer and recombination as drivers of diversity in this swine pathogen and zoonotic agent.

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July 7, 2019  |  

The identification of novel diagnostic marker genes for the detection of beer spoiling Pediococcus damnosus strains using the BlAst Diagnostic Gene findEr.

As the number of bacterial genomes increases dramatically, the demand for easy to use tools with transparent functionality and comprehensible output for applied comparative genomics grows as well. We present BlAst Diagnostic Gene findEr (BADGE), a tool for the rapid prediction of diagnostic marker genes (DMGs) for the differentiation of bacterial groups (e.g. pathogenic / nonpathogenic). DMG identification settings can be modified easily and installing and running BADGE does not require specific bioinformatics skills. During the BADGE run the user is informed step by step about the DMG finding process, thus making it easy to evaluate the impact of chosen settings and options. On the basis of an example with relevance for beer brewing, being one of the oldest biotechnological processes known, we show a straightforward procedure, from phenotyping, genome sequencing, assembly and annotation, up to a discriminant marker gene PCR assay, making comparative genomics a means to an end. The value and the functionality of BADGE were thoroughly examined, resulting in the successful identification and validation of an outstanding novel DMG (fabZ) for the discrimination of harmless and harmful contaminations of Pediococcus damnosus, which can be applied for spoilage risk determination in breweries. Concomitantly, we present and compare five complete P. damnosus genomes sequenced in this study, finding that the ability to produce the unwanted, spoilage associated off-flavor diacetyl is a plasmid encoded trait in this important beer spoiling species.

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July 7, 2019  |  

Filling in the gap of human chromosome 4: Single Molecule Real Time sequencing of macrosatellite repeats in the facioscapulohumeral muscular dystrophy locus.

A majority of facioscapulohumeral muscular dystrophy (FSHD) is caused by contraction of macrosatellite repeats called D4Z4 that are located in the subtelomeric region of human chromosome 4q35. Sequencing the FSHD locus has been technically challenging due to its long size and nearly identical nature of repeat elements. Here we report sequencing and partial assembly of a BAC clone carrying an entire FSHD locus by a single molecule real time (SMRT) sequencing technology which could produce long reads up to about 18 kb containing D4Z4 repeats. De novo assembly by Hierarchical Genome Assembly Process 1 (HGAP.1) yielded a contig of 41 kb containing all but a part of the most distal D4Z4 element. The validity of the sequence model was confirmed by an independent approach employing anchored multiple sequence alignment by Kalign using reads containing unique flanking sequences. Our data will provide a basis for further optimization of sequencing and assembly conditions of D4Z4.

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July 7, 2019  |  

Stability of the encoding plasmids and surface expression of CS6 differs in enterotoxigenic Escherichia coli (ETEC) encoding Different heat-stable (ST) enterotoxins (STh and STp).

Enterotoxigenic Escherichia coli (ETEC), one of the most common reasons of diarrhea among infants and children in developing countries, causes disease by expression of either or both of the enterotoxins heat-labile (LT) and heat-stable (ST; divided into human-type [STh] and porcine-type [STp] variants), and colonization factors (CFs) among which CS6 is one of the most prevalent ETEC CFs. In this study we show that ETEC isolates expressing CS6+STh have higher copy numbers of the cssABCD operon encoding CS6 than those expressing CS6+STp. Long term cultivation of up to ten over-night passages of ETEC isolates harboring CS6+STh (n = 10) or CS6+STp (n = 15) showed instability of phenotypic expression of CS6 in a majority of the CS6+STp isolates, whereas most of the CS6+STh isolates retained CS6 expression. The observed instability was a correlated with loss of genes cssA and cssD as examined by PCR. Mobilization of the CS6 plasmid from an unstable CS6+STp isolate into a laboratory E. coli strain resulted in loss of the plasmid after a single over-night passage whereas the plasmid from an CS6+STh strain was retained in the laboratory strain during 10 passages. A sequence comparison between the CS6 plasmids from a stable and an unstable ETEC isolate revealed that genes necessary for plasmid stabilization, for example pemI, pemK, stbA, stbB and parM, were not present in the unstable ETEC isolate. Our results indicate that stable retention of CS6 may in part be affected by the stability of the plasmid on which both CS6 and STp or STh are located.

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