Zhengyuan Wang

Comparative and demographic analysis of orang-utan genomes

‘Orang-utan’ is derived from a Malay term meaning ‘man of the forest’ and aptly describes the southeast Asian great apes native to Sumatra and Borneo. The orang-utan species, Pongo abelii (Sumatran) and Pongo pygmaeus (Bornean), are the most phylogenetically distant great apes from humans, thereby providing an informative perspective on hominid evolution. Here we present a Sumatran orang-utan draft genome assembly and short read sequence data from five Sumatran and five Bornean orang-utan genomes. Our analyses reveal that, compared to other primates, the orang-utan genome has many unique features. Structural evolution of the orang-utan genome has proceeded much more slowly than other great apes, evidenced by fewer rearrangements, less segmental duplication, a lower rate of gene family turnover and surprisingly quiescent Alu repeats, which have played a major role in restructuring other primate genomes. We also describe a primate polymorphic neocentromere, found in both Pongo species, emphasizing the gradual evolution of orang-utan genome structure. Orang-utans have extremely low energy usage for a eutherian mammal, far lower than their hominid relatives. Adding their genome to the repertoire of sequenced primates illuminates new signals of positive selection in several pathways including glycolipid metabolism. From the population perspective, both Pongo species are deeply diverse; however, Sumatran individuals possess greater diversity than their Bornean counterparts, and more species-specific variation. Our estimate of Bornean/Sumatran speciation time, 400,000 years ago, is more recent than most previous studies and underscores the complexity of the orang-utan speciation process. Despite a smaller modern census population size, the Sumatran effective population size (Ne) expanded exponentially relative to the ancestral Ne after the split, while Bornean Ne declined over the same period. Overall, the resources and analyses presented here offer new opportunities in evolutionary genomics, insights into hominid biology, and an extensive database of variation for conservation efforts.

The draft genome of the parasitic nematode Trichinella spiralis

Genome evolution studies for the phylum Nematoda have been limited by focusing on comparisons involving Caenorhabditis elegans. We report a draft genome sequence of Trichinella spiralis, a food-borne zoonotic parasite, which is the most common cause of human trichinellosis. This parasitic nematode is an extant member of a clade that diverged early in the evolution of the phylum, enabling identification of archetypical genes and molecular signatures exclusive to nematodes. We sequenced the 64-Mb nuclear genome, which is estimated to contain 15,808 protein-coding genes, at ∼35-fold coverage using whole-genome shotgun and hierarchal map–assisted sequencing. Comparative genome analyses support intrachromosomal rearrangements across the phylum, disproportionate numbers of protein family deaths over births in parasitic compared to a non-parasitic nematode and a preponderance of gene-loss and -gain events in nematodes relative to Drosophila melanogaster. This genome sequence and the identified pan-phylum characteristics will contribute to genome evolution studies of Nematoda as well as strategies to combat global parasites of humans, food animals and crops.

Nematode.net update 2008: improvements enabling more efficient data mining and comparative nematode genomics

Nematode.net (http://nematode.net) is a publicly available resource dedicated to the study of parasitic nematodes. In 2000, the Genome Center at Washington University (GC) joined a consortium including the Nematode Genomics group in Edinburgh, and the Pathogen Sequencing Unit of the Sanger Institute to generate expressed sequence tags (ESTs) as an inexpensive and efficient solution for gene discovery in parasitic nematodes. As of 2008 the GC, sampling key parasites of humans, animals and plants, has generated over 500 000 ESTs and 1.2 million genome survey sequences from more than 30 non-Caenorhabditis elegans nematodes. Nematode.net was implemented to offer user-friendly access to data produced by this project. In addition to sequence data, the site hosts: assembled NemaGene clusters in GBrowse views characterizing composition and protein homology, functional Gene Ontology annotations presented via the AmiGO browser, KEGG-based graphical display of NemaGene clusters mapped to metabolic pathways, codon usage tables, NemFam protein families which represent conserved nematode-restricted coding sequences not found in public protein databases, a web-based WU-BLAST search tool that allows complex querying and other assorted resources. The primary aim of Nematode.net is the dissemination of this diverse collection of information to the broader scientific community in a way that is useful, consistent, centralized and enduring.

Characterizing Ancylostoma caninum transcriptome and exploring nematode parasitic adaptation

BackgroundHookworm infection is one of the most important neglected diseases in developing countries, with approximately 1 billion people infected worldwide. To better understand hookworm biology and nematode parasitism, the present study generated a near complete transcriptome of the canine hookworm Ancylostoma caninum to a very high coverage using high throughput technology, and compared it to those of the free-living nematode Caenorhabditis elegans and the parasite Brugia malayi.ResultsThe generated transcripts from four developmental stages, infective L3, serum stimulated L3, adult male and adult female, covered 93% of the A. caninum transcriptome. The broad diversity among nematode transcriptomes was confirmed, and an impact of parasitic adaptation on transcriptome diversity was inferred. Intra-population analysis showed that A. caninum has higher coding sequence diversity than humans. Examining the developmental expression profiles of A. caninum revealed major transitions in gene expression from larval stages to adult. Adult males expressed the highest number of selectively expressed genes, but adult female expressed the highest number of selective parasitism-related genes. Genes related to parasitism adaptation and A. caninum specific genes exhibited more expression selectivity while those conserved in nematodes tend to be consistently expressed. Parasitism related genes were expressed more selectively in adult male and female worms. The comprehensive analysis of digital expression profiles along with transcriptome comparisons enabled identification of a set of parasitism genes encoding secretory proteins in animal parasitic nematode.ConclusionsThis study validated the usage of deep sequencing for gene expression profiling. Parasitic adaptation of the canine hookworm is related to its diversity and developmental dynamics. This comprehensive comparative genomic and expression study substantially improves our understanding of the basic biology and parasitism of hookworms and, is expected, in the long run, to accelerate research toward development of vaccines and novel anthelmintics.

Optimizing Read Mapping to Reference Genomes to Determine Composition and Species Prevalence in Microbial Communities

The Human Microbiome Project (HMP) aims to characterize the microbial communities of 18 body sites from healthy individuals. To accomplish this, the HMP generated two types of shotgun data: reference shotgun sequences isolated from different anatomical sites on the human body and shotgun metagenomic sequences from the microbial communities of each site. The alignment strategy for characterizing these metagenomic communities using available reference sequence is important to the success of HMP data analysis. Six next-generation aligners were used to align a community of known composition against a database comprising reference organisms known to be present in that community. All aligners report nearly complete genome coverage (>97%) for strains with over 6X depth of coverage, however they differ in speed, memory requirement and ease of use issues such as database size limitations and supported mapping strategies. The selected aligner was tested across a range of parameters to maximize sensitivity while maintaining a low false positive rate. We found that constraining alignment length had more impact on sensitivity than does constraining similarity in all cases tested. However, when reference species were replaced with phylogenetic neighbors, similarity begins to play a larger role in detection. We also show that choosing the top hit randomly when multiple, equally strong mappings are available increases overall sensitivity at the expense of taxonomic resolution. The results of this study identified a strategy that was used to map over 3 tera-bases of microbial sequence against a database of more than 5,000 reference genomes in just over a month.

NemaPath: online exploration of KEGG-based metabolic pathways for nematodes

BackgroundNematode.net http://www.nematode.net is a web-accessible resource for investigating gene sequences from parasitic and free-living nematode genomes. Beyond the well-characterized model nematode C. elegans, over 500,000 expressed sequence tags (ESTs) and nearly 600,000 genome survey sequences (GSSs) have been generated from 36 nematode species as part of the Parasitic Nematode Genomics Program undertaken by the Genome Center at Washington University School of Medicine. However, these sequencing data are not present in most publicly available protein databases, which only include sequences in Swiss-Prot. Swiss-Prot, in turn, relies on GenBank/Embl/DDJP for predicted proteins from complete genomes or full-length proteins.DescriptionHere we present the NemaPath pathway server, a web-based pathway-level visualization tool for navigating putative metabolic pathways for over 30 nematode species, including 27 parasites. The NemaPath approach consists of two parts: 1) a backend tool to align and evaluate nematode genomic sequences (curated EST contigs) against the annotated Kyoto Encyclopedia of Genes and Genomes (KEGG) protein database; 2) a web viewing application that displays annotated KEGG pathway maps based on desired confidence levels of primary sequence similarity as defined by a user. NemaPath also provides cross-referenced access to nematode genome information provided by other tools available on Nematode.net, including: detailed NemaGene EST cluster information; putative translations; GBrowse EST cluster views; links from nematode data to external databases for corresponding synonymous C. elegans counterparts, subject matches in KEGGs gene database, and also KEGG Ontology (KO) identification.ConclusionThe NemaPath server hosts metabolic pathway mappings for 30 nematode species and is available on the World Wide Web at http://nematode.net/cgi-bin/keggview.cgi. The nematode source sequences used for the metabolic pathway mappings are available via FTP http://www.nematode.net/FTP/index.php, as provided by the Genome Center at Washington University School of Medicine.

Transcription profiling reveals stage- and function-dependent expression patterns in the filarial nematode Brugia malayi

BackgroundBrugia malayi is a nematode parasite that causes lymphatic filariasis, a disfiguring and disabiling tropical disease. Although a first draft genome sequence was released in 2007, very little is understood about transcription programs that govern developmental changes required for the parasite’s development and survival in its mammalian and insect hosts.ResultsWe used a microarray with probes that represent some 85% of predicted genes to generate gene expression profiles for seven parasite life cycle stages/sexes. Approximately 41% of transcripts with detectable expression signals were differentially expressed across lifecycle stages. Twenty-six percent of transcripts were exclusively expressed in a single parasite stage, and 27% were expressed in all stages studied. K-means clustering of differentially expressed transcripts revealed five major transcription patterns that were associated with parasite lifecycle stages or gender. Examination of known stage-associated transcripts validated these data sets and suggested that newly identified stage or gender-associated transcripts may exercise biological functions in development and reproduction. The results also indicate that genes with similar transcription patterns were often involved in similar functions or cellular processes. For example, nuclear receptor family gene transcripts were upregulated in gene expression pattern four (female-enriched) while protein kinase gene family transcripts were upregulated in expression pattern five (male-enriched). We also used pair-wise comparisons to identify transcriptional changes between life cycle stages and sexes.ConclusionsAnalysis of gene expression patterns of lifecycle in B. malayi has provided novel insights into the biology of filarial parasites. Proteins encoded by stage-associated and/or stage-specific transcripts are likely to be critically important for key parasite functions such as establishment and maintenance of infection, development, reproduction, and survival in the host. Some of these may be useful targets for vaccines or new drug treatments for filariasis.

Systematic analysis of insertions and deletions specific to nematode proteins and their proposed functional and evolutionary relevance

BackgroundAmino acid insertions and deletions in proteins are considered relatively rare events, and their associations with the evolution and adaptation of organisms are not yet understood. In this study, we undertook a systematic analysis of over 214,000 polypeptides from 32 nematode species and identified insertions and deletions unique to nematode proteins in more than 1000 families and provided indirect evidence that these alterations are linked to the evolution and adaptation of nematodes.ResultsAmino acid alterations in sequences of nematodes were identified by comparison with homologous sequences from a wide range of eukaryotic (metzoan) organisms. This comparison revealed that the proteins inferred from transcriptomic datasets for nematodes contained more deletions than insertions, and that the deletions tended to be larger in length than insertions, indicating a decreased size of the transcriptome of nematodes compared with other organisms. The present findings showed that this reduction is more pronounced in parasitic nematodes compared with the free-living nematodes of the genus Caenorhabditis. Consistent with a requirement for conservation in proteins involved in the processing of genetic information, fewer insertions and deletions were detected in such proteins. On the other hand, more insertions and deletions were recorded for proteins inferred to be involved in the endocrine and immune systems, suggesting a link with adaptation. Similarly, proteins involved in multiple cellular pathways tended to display more deletions and insertions than those involved in a single pathway. The number of insertions and deletions shared by a range of plant parasitic nematodes were higher for proteins involved in lipid metabolism and electron transport compared with other nematodes, suggesting an association between metabolic adaptation and parasitism in plant hosts. We also identified three sizable deletions from proteins found to be specific to and shared by parasitic nematodes, which, given their uniqueness, might serve as target candidates for drug design.ConclusionThis study illustrates the significance of using comparative genomics approaches to identify molecular elements unique to parasitic nematodes, which have adapted to a particular host organism and mode of existence during evolution. While the focus of this study was on nematodes, the approach has applicability to a wide range of other groups of organisms.

Targeting protein-protein interactions for parasite control.

Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific ortholgous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable.

Identification of hookworm DAF-16/FOXO response elements and direct gene targets.

Background The infective stage of the parasitic nematode hookworm is developmentally arrested in the environment and needs to infect a specific host to complete its life cycle. The canine hookworm (Ancylostoma caninum) is an excellent model for investigating human hookworm infections. The transcription factor of A. caninum, Ac-DAF-16, which has a characteristic fork head or “winged helix” DNA binding domain (DBD), has been implicated in the resumption of hookworm development in the host. However, the precise roles of Ac-DAF-16 in hookworm parasitism and its downstream targets are unknown. In the present study, we combined molecular techniques and bioinformatics to identify a group of Ac-DAF-16 binding sites and target genes. Methodology/Principal Findings The DNA binding domain of Ac-DAF-16 was used to select genomic fragments by in vitro genomic selection. Twenty four bound genomic fragments were analyzed for the presence of the DAF-16 family binding element (DBE) and possible alternative Ac-DAF-16 bind motifs. The 22 genes linked to these genomic fragments were identified using bioinformatics tools and defined as candidate direct gene targets of Ac-DAF-16. Their developmental stage-specific expression patterns were examined. Also, a new putative DAF-16 binding element was identified. Conclusions/Significance Our results show that Ac-DAF-16 is involved in diverse biological processes throughout hookworm development. Further investigation of these target genes will provide insights into the molecular basis by which Ac-DAF-16 regulates its downstream gene network in hookworm infection.