Man Kit Cheung

Composition and genetic diversity of picoeukaryotes in subtropical coastal waters as revealed by 454 pyrosequencing

Information on genetic diversity of picoeukaryotes (<2–3 μm) comes mainly from traditional gene cloning and sequencing, but this method suffers from cloning biases and limited throughput. In this study, we explored the feasibility of using the cloning-independent and massively parallel 454 pyrosequencing technology to study the composition and genetic diversity of picoeukaryotes in the coastal waters of the subtropical western Pacific using the hypervariable V4 region of the 18S rRNA gene. Picoeukaryote assemblages between two sites with different hydrography and trophic status were also compared. The approach gave a high coverage of the community at genetic difference ⩾5% but still underestimated the total diversity at a genetic difference ⩽2%. Diversity of picoeukaryotes was higher in an oligomesotrophic bay than in a eutrophic bay. Stramenopiles, dinoflagellates, ciliates and prasinophytes were the dominant groups comprising approximately 27, 19, 11 and 11%, respectively, of the picoeukaryotes. Water samples collected from the two bays contained different high-level taxonomic groups and phylotype operational taxonomic units of picoeukaryotes. Our study represents one of the first and most comprehensive examinations of marine picoeukaryotic diversity using the 454 sequencing-by-synthesis technology.

BSRD: a repository for bacterial small regulatory RNA

In bacteria, small regulatory non-coding RNAs (sRNAs) are the most abundant class of post-transcriptional regulators. They are involved in diverse processes including quorum sensing, stress response, virulence and carbon metabolism. Recent developments in high-throughput techniques, such as genomic tiling arrays and RNA-Seq, have allowed efficient detection and characterization of bacterial sRNAs. However, a comprehensive repository to host sRNAs and their annotations is not available. Existing databases suffer from a limited number of bacterial species or sRNAs included. In addition, these databases do not have tools to integrate or analyse high-throughput sequencing data. Here, we have developed BSRD (http://kwanlab.bio.cuhk.edu.hk/BSRD), a comprehensive bacterial sRNAs database, as a repository for published bacterial sRNA sequences with annotations and expression profiles. BSRD contains over nine times more experimentally validated sRNAs than any other available databases. BSRD also provides combinatorial regulatory networks of transcription factors and sRNAs with their common targets. We have built and implemented in BSRD a novel RNA-Seq analysis platform, sRNADeep, to characterize sRNAs in large-scale transcriptome sequencing projects. We will update BSRD regularly.

Sputum Microbiota in Tuberculosis as Revealed by 16S rRNA Pyrosequencing

Background Tuberculosis (TB) remains a global threat in the 21st century. Traditional studies of the disease are focused on the single pathogen Mycobacterium tuberculosis. Recent studies have revealed associations of some diseases with an imbalance in the microbial community. Characterization of the TB microbiota could allow a better understanding of the disease. Methodology/Principal Findings Here, the sputum microbiota in TB infection was examined by using 16S rRNA pyrosequencing. A total of 829,873 high-quality sequencing reads were generated from 22 TB and 14 control sputum samples. Firmicutes, Proteobacteria, Bacteroidetes, Actinobacteria, and Fusobacteria were the five major bacterial phyla recovered, which together composed over 98% of the microbial community. Proteobacteria and Bacteroidetes were more represented in the TB samples and Firmicutes was more predominant in the controls. Sixteen major bacterial genera were recovered. Streptococcus, Neisseria and Prevotella were the most predominant genera, which were dominated by several operational taxonomic units grouped at a 97% similarity level. Actinomyces, Fusobacterium, Leptotrichia, Prevotella, Streptococcus, and Veillonella were found in all TB samples, possibly representing the core genera in TB sputum microbiota. The less represented genera Mogibacterium, Moryella and Oribacterium were enriched statistically in the TB samples, while a genus belonging to the unclassified Lactobacillales was enriched in the controls. The diversity of microbiota was similar in the TB and control samples. Conclusions/Significance The composition and diversity of sputum microbiota in TB infection was characterized for the first time by using high-throughput pyrosequencing. It lays the framework for examination of potential roles played by the diverse microbiota in TB pathogenesis and progression, and could ultimately facilitate advances in TB treatment.

2011 German Escherichia coli O104:H4 outbreak: whole-genome phylogeny without alignment

BackgroundA large-scale Escherichia coli O104:H4 outbreak occurred in Germany from May to July 2011, causing numerous cases of hemolytic-uremic syndrome (HUS) and deaths. Genomes of ten outbreak isolates and a historical O104:H4 strain isolated in 2001 were sequenced using different new generation sequencing platforms. Phylogenetic analyses were performed using various approaches which either are not genome-wide or may be subject to errors due to poor sequence alignment. Also, detailed pathogenicity analyses on the 2001 strain were not available.FindingsWe reconstructed the phylogeny of E. coli using the genome-wide and alignment-free feature frequency profile method and revealed the 2001 strain to be the closest relative to the 2011 outbreak strain among all available E. coli strains at present and confirmed findings from previous alignment-based phylogenetic studies that the HUS-causing O104:H4 strains are more closely related to typical enteroaggregative E. coli (EAEC) than to enterohemorrhagic E. coli. Detailed re-examination of pathogenicity-related virulence factors and secreted proteins showed that the 2001 strain possesses virulence factors shared between typical EAEC and the 2011 outbreak strain.ConclusionsOur study represents the first attempt to elucidate the whole-genome phylogeny of the 2011 German outbreak using an alignment-free method, and suggested a direct line of ancestry leading from a putative EAEC-like ancestor through the 2001 strain to the 2011 outbreak strain.

Human proteins with target sites of multiple post-translational modification types are more prone to be involved in disease.

Many proteins can be modified by multiple types of post-translational modifications (Mtp-proteins). Although some post-translational modifications (PTMs) have recently been found to be associated with life-threatening diseases like cancers and neurodegenerative disorders, the underlying mechanisms remain enigmatic to date. In this study, we examined the relationship of human Mtp-proteins and disease and systematically characterized features of these proteins. Our results indicated that Mtp-proteins are significantly more inclined to participate in disease than proteins carrying no known PTM sites. Mtp-proteins were found significantly enriched in protein complexes, having more protein partners and preferred to act as hubs/superhubs in protein-protein interaction (PPI) networks. They possess a distinct functional focus, such as chromatin assembly or disassembly, and reside in biased, multiple subcellular localizations. Moreover, most Mtp-proteins harbor more intrinsically disordered regions than the others. Mtp-proteins carrying PTM types biased toward locating in the ordered regions were mainly related to protein-DNA complex assembly. Examination of the energetic effects of PTMs on the stability of PPI revealed that only a small fraction of single PTM events influence the binding energy of >2 kcal/mol, whereas the binding energy can change dramatically by combinations of multiple PTM types. Our work not only expands the understanding of Mtp-proteins but also discloses the potential ability of Mtp-proteins to act as key elements in disease development.

Microbial diversity in shallow-water hydrothermal sediments of Kueishan Island, Taiwan as revealed by pyrosequencing.

Kueishan Island is a young volcanic island in the southernmost edge of the Okinawa Trough in the northeastern part of Taiwan. A cluster of hydrothermal vents is located off the southeastern tip of the Island at water depths between 10 and 80 m. This paper presents the results of the first study on the microbial communities in bottom sediments collected from the shallow‐water hydrothermal vents of Kueishan Island. Small‐subunit ribosomal RNA gene‐based high‐throughput 454 pyrosequencing was used to characterize the assemblages of bacteria, archaea, and small eukaryotes in sediment samples collected at various distances from the hydrothermal vents. Sediment from the vent area contained the highest diversity of archaea and the lowest diversity of bacteria and small eukaryotes. Epsilonproteobacteria were the most abundant group in the vent sediment, but their abundance decreased with increasing distance from the vent area. Most Epsilonproteobacteria belonged to the mesophilic chemolithoautotrophic genera Sulfurovum and Sulfurimonas. Recent reports on these two genera have come from deep‐sea hydrothermal vents. Conversely, the relative contribution of Gammaproteobacteria to the bacterial community increased with increasing distance from the vent area. Our study revealed the contrasting effects of venting on the benthic bacterial and archaeal communities, and showed that the sediments of the shallow‐waters hydrothermal vents were dominated by chemoautotrophic bacteria. The present work broadens our knowledge on microbial diversity in shallow‐water hydrothermal vent habitats.

Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes

BackgroundGenetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.FindingsShiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.ConclusionsWe used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.

Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

BackgroundVibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.ResultsHere we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.ConclusionsWe have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

High-Density Transcriptional Initiation Signals Underline Genomic Islands in Bacteria

Genomic islands (GIs), frequently associated with the pathogenicity of bacteria and having a substantial influence on bacterial evolution, are groups of “alien” elements which probably undergo special temporal–spatial regulation in the host genome. Are there particular hallmark transcriptional signals for these “exotic” regions? We here explore the potential transcriptional signals that underline the GIs beyond the conventional views on basic sequence composition, such as codon usage and GC property bias. It showed that there is a significant enrichment of the transcription start positions (TSPs) in the GI regions compared to the whole genome of Salmonella enterica and Escherichia coli. There was up to a four-fold increase for the 70% GIs, implying high-density TSPs profile can potentially differentiate the GI regions. Based on this feature, we developed a new sliding window method GIST, Genomic-island Identification by Signals of Transcription, to identify these regions. Subsequently, we compared the known GI-associated features of the GIs detected by GIST and by the existing method Islandviewer to those of the whole genome. Our method demonstrates high sensitivity in detecting GIs harboring genes with biased GI-like function, preferred subcellular localization, skewed GC property, shorter gene length and biased “non-optimal” codon usage. The special transcriptional signals discovered here may contribute to the coordinate expression regulation of foreign genes. Finally, by using GIST, we detected many interesting GIs in the 2011 German E. coli O104:H4 outbreak strain TY-2482, including the microcin H47 system and gene cluster ycgXEFZ-ymgABC that activates the production of biofilm matrix. The aforesaid findings highlight the power of GIST to predict GIs with distinct intrinsic features to the genome. The heterogeneity of cumulative TSPs profiles may not only be a better identity for “alien” regions, but also provide hints to the special evolutionary course and transcriptional regulation of GI regions.

Population genomic analysis uncovers environmental stress-driven selection and adaptation of Lentinula edodes population in China

The elucidation of genome-wide variations could help reveal aspects of divergence, domestication, and adaptation of edible mushrooms. Here, we resequenced the whole genomes of 39 wild and 21 cultivated strains of Chinese Lentinula edodes, the shiitake mushroom. We identified three distinct genetic groups in the Chinese L. edodes population with robust differentiation. Results of phylogenetic and population structure analyses suggest that the cultivated strains and most of the wild trains of L. edodes in China possess different gene pools and two outlier strains show signatures of hybridization between groups. Eighty-four candidate genes contributing to population divergence were detected in outlier analysis, 18 of which are involved in response to environmental stresses. Gene enrichment analysis of group-specific single nucleotide polymorphisms showed that the cultivated strains were genetically diversified in biological processes related to stress response. As the formation of fruiting bodies is a stress-response process, we postulate that environment factors, such as temperature, drove the population divergence of L. edodes in China by natural or artificial selection. We also found phenotypic variations between groups and identified some wild strains that have potential to diversify the genetic pool for improving agricultural traits of L. edodes cultivars in China.