Genome-wide subcellular protein map for the flagellate parasite Trypanosoma brucei.

Trypanosoma brucei is a model trypanosomatid, an important group of human, animal and plant unicellular parasites. Understanding their complex cell architecture and life cycle is challenging because, as with most eukaryotic microbes, ~50% of genome-encoded proteins have completely unknown functions. Here, using fluorescence microscopy and cell lines expressing endogenously tagged proteins, we mapped the subcellular localization of 89% of the T. brucei proteome, a resource we call TrypTag. We provide clues to function and define lineage-specific organelle adaptations for parasitism, mapping the ultraconserved cellular architecture of eukaryotes, including the first comprehensive 'cartographic' analysis of the eukaryotic flagellum, which is vital for morphogenesis and pathology. To demonstrate the power of this resource, we identify novel organelle subdomains and changes in molecular composition through the cell cycle. TrypTag is a transformative resource, important for hypothesis generation for both eukaryotic evolutionary molecular cell biology and fundamental parasite cell biology.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

Genome Assemblies across the Diverse Evolutionary Spectrum of Leishmania Protozoan Parasites.

We report the high-quality draft assemblies and gene annotations for 13 species and/or strains of the protozoan parasite genera Leishmania, Endotrypanum, and Crithidia, which span the phylogenetic diversity of the subfamily Leishmaniinae within the kinetoplastid order of the phylum Euglenazoa. These resources will support studies on the origins of parasitism.

High Levels of Genetic Diversity within Nilo-Saharan Populations: Implications for Human Adaptation.

Africa contains more human genetic variation than any other continent, but the majority of the population-scale analyses of the African peoples have focused on just two of the four major linguistic groups, the Niger-Congo and Afro-Asiatic, leaving the Nilo-Saharan and Khoisan populations under-represented. In order to assess genetic variation and signatures of selection within a Nilo-Saharan population and between the Nilo-Saharan and Niger-Congo and Afro-Asiatic, we sequenced 50 genomes from the Nilo-Saharan Lugbara population of North-West Uganda and 250 genomes from 6 previously unsequenced Niger-Congo populations. We compared these data to data from a further 16 Eurasian and African populations including the Gumuz, another putative Nilo-Saharan population from Ethiopia. Of the 21 million variants identified in the Nilo-Saharan population, 3.57 million (17%) were not represented in dbSNP and included predicted non-synonymous mutations with possible phenotypic effects. We found greater genetic differentiation between the Nilo-Saharan Lugbara and Gumuz populations than between any two Afro-Asiatic or Niger-Congo populations. F3 tests showed that Gumuz contributed a genetic component to most Niger-Congo B populations whereas Lugabara did not. We scanned the genomes of the Lugbara for evidence of selective sweeps. We found selective sweeps at four loci (SLC24A5, SNX13, TYRP1, and UVRAG) associated with skin pigmentation, three of which already have been reported to be under selection. These selective sweeps point toward adaptations to the intense UV radiation of the Sahel.

Targeted Next-Generation Sequencing of 117 Routine Clinical Samples Provides Further Insights into the Molecular Landscape of Uveal Melanoma.

Uveal melanoma (UM) has well-characterised somatic copy number alterations (SCNA) in chromosomes 1, 3, 6 and 8, in addition to mutations in GNAQ, GNA11, CYSLTR2, PLCB4, BAP1, SF3B1 and EIF1AX, most being linked to metastatic-risk. To gain further insight into the molecular landscape of UM, we designed a targeted next-generation sequencing (NGS) panel to detect SCNA and mutations in routine clinical UM samples. We compared hybrid-capture and amplicon-based target enrichment methods and tested a larger cohort of primary UM samples on the best performing panel. UM clinical samples processed either as fresh-frozen, formalin-fixed paraffin embedded (FFPE), small intraocular biopsies or following irradiation were successfully profiled using NGS, with hybrid capture outperforming the PCR-based enrichment methodology. We identified monosomy 3 (M3)-UM that were wild-type for BAP1 but harbored SF3B1 mutations, novel frameshift deletions in SF3B1 and EIF1AX, as well as a PLCB4 mutation outside of the hotspot on exon 20 coinciding with a GNAQ mutation in some UM. We observed samples that harboured mutations in both BAP1 and SF3B1, and SF3B1 and EIF1AX, respectively. Novel mutations were also identified in TTC28, KTN1, CSMD1 and TP53BP1. NGS can simultaneously assess SCNA and mutation data in UM, in a reliable and reproducible way, irrespective of sample type or previous processing. BAP1 and SF3B1 mutations, in addition to 8q copy number, are of added importance when determining UM patient outcome.

Copy number variation in human genomes from three major ethno-linguistic groups in Africa.

BACKGROUND: Copy number variation is an important class of genomic variation that has been reported in 75% of the human genome. However, it is underreported in African populations. Copy number variants (CNVs) could have important impacts on disease susceptibility and environmental adaptation. To describe CNVs and their possible impacts in Africans, we sequenced genomes of 232 individuals from three major African ethno-linguistic groups: (1) Niger Congo A from Guinea and Côte d'Ivoire, (2) Niger Congo B from Uganda and the Democratic Republic of Congo and (3) Nilo-Saharans from Uganda. We used GenomeSTRiP and cn.MOPS to identify copy number variant regions (CNVRs). RESULTS: We detected 7608 CNVRs, of which 2172 were only deletions, 2384 were only insertions and 3052 had both. We detected 224 previously un-described CNVRs. The majority of novel CNVRs were present at low frequency and were not shared between populations. We tested for evidence of selection associated with CNVs and also for population structure. Signatures of selection identified previously, using SNPs from the same populations, were overrepresented in CNVRs. When CNVs were tagged with SNP haplotypes to identify SNPs that could predict the presence of CNVs, we identified haplotypes tagging 3096 CNVRs, 372 CNVRs had SNPs with evidence of selection (iHS > 3) and 222 CNVRs had both. This was more than expected (p < 0.0001) and included loci where CNVs have previously been associated with HIV, Rhesus D and preeclampsia. When integrated with 1000 Genomes CNV data, we replicated their observation of population stratification by continent but no clustering by populations within Africa, despite inclusion of Nilo-Saharans and Niger-Congo populations within our dataset. CONCLUSIONS: Novel CNVRs in the current study increase representation of African diversity in the database of genomic variants. Over-representation of CNVRs in SNP signatures of selection and an excess of SNPs that both tag CNVs and are subject to selection show that CNVs may be the actual targets of selection at some loci. However, unlike SNPs, CNVs alone do not resolve African ethno-linguistic groups. Tag haplotypes for CNVs identified may be useful in predicting African CNVs in future studies where only SNP data is available.

Increasing prevalence of a fluoroquinolone resistance mutation amongst Campylobacter jejuni isolates from four human infectious intestinal disease studies in the United Kingdom.

BACKGROUND: Campylobacter jejuni is the most common bacterial cause of human infectious intestinal disease. METHODS: We genome sequenced 601 human C. jejuni isolates, obtained from two large prospective studies of infectious intestinal disease (IID1 [isolates from 1993-1996; n = 293] and IID2 [isolates from 2008-2009; n = 93]), the INTEGRATE project [isolates from 2016-2017; n = 52] and the ENIGMA project [isolates from 2017; n = 163]. RESULTS: There was a significant increase in the prevalence of the T86I mutation conferring resistance to fluoroquinolone between each of the three later studies (IID2, INTEGRATE and ENIGMA) and IID1. Although the distribution of major multilocus sequence types (STs) was similar between the studies, there were changes in both the abundance of minority STs associated with the T86I mutation, and the abundance of clones within single STs associated with the T86I mutation. DISCUSSION: Four population-based studies of community diarrhoea over a 25 year period revealed an increase over time in the prevalence of the T86I amongst isolates of C. jejuni associated with human gastrointestinal disease in the UK. Although associated with many STs, much of the increase is due to the expansion of clones associated with the resistance mutation.

Application of long read sequencing to determine expressed antigen diversity in Trypanosoma brucei infections.

Antigenic variation is employed by many pathogens to evade the host immune response, and Trypanosoma brucei has evolved a complex system to achieve this phenotype, involving sequential use of variant surface glycoprotein (VSG) genes encoded from a large repertoire of ~2,000 genes. T. brucei express multiple, sometimes closely related, VSGs in a population at any one time, and the ability to resolve and analyse this diversity has been limited. We applied long read sequencing (PacBio) to VSG amplicons generated from blood extracted from batches of mice sacrificed at time points (days 3, 6, 10 and 12) post-infection with T. brucei TREU927. The data showed that long read sequencing is reliable for resolving variant differences between VSGs, and demonstrated that there is significant expressed diversity (449 VSGs detected across 20 mice) and across the timeframe of study there was a clear semi-reproducible pattern of expressed diversity (median of 27 VSGs per sample at day 3 post infection (p.i.), 82 VSGs at day 6 p.i., 187 VSGs at day 10 p.i. and 132 VSGs by day 12 p.i.). There was also consistent detection of one VSG dominating expression across replicates at days 3 and 6, and emergence of a second dominant VSG across replicates by day 12. The innovative application of ecological diversity analysis to VSG reads enabled characterisation of hierarchical VSG expression in the dataset, and resulted in a novel method for analysing such patterns of variation. Additionally, the long read approach allowed detection of mosaic VSG expression from very few reads-the earliest in infection that such events have been detected. Therefore, our results indicate that long read analysis is a reliable tool for resolving diverse gene expression profiles, and provides novel insights into the complexity and nature of VSG expression in trypanosomes, revealing significantly higher diversity than previously shown and the ability to identify mosaic gene formation early during the infection process.

Association between IL1 gene polymorphism and human African trypanosomiasis in populations of sleeping sickness foci of southern Cameroon.

BACKGROUND: Human African Trypanosomiasis (HAT) is a neglected tropical disease caused by infections due to Trypanosoma brucei subspecies. In addition to the well-established environmental and behavioural risks of becoming infected, there is evidence for a genetic component to the response to trypanosome infection. We undertook a candidate gene case-control study to investigate genetic associations further. METHODOLOGY: We genotyped one polymorphism in each of seven genes (IL1A, IL1RN, IL4RN, IL6, HP, HPR, and HLA-G) in 73 cases and 250 controls collected from 19 ethno-linguistic subgroups stratified into three major ethno-linguistic groups, 2 pooled ethno-linguistic groups and 11 ethno-linguistic subgroups from three Cameroonian HAT foci. The seven polymorphic loci tested consisted of three SNPs, three variable numbers of tandem repeat (VNTR) and one INDEL. RESULTS: We found that the genotype (TT) and minor allele (T) of IL1A gene as well as the genotype 1A3A of IL1RN were associated with an increased risk of getting Trypanosoma brucei gambiense and develop HAT when all data were analysed together and also when stratified by the three major ethno-linguistic groups, 2 pooled ethno-linguistic subgroups and 11 ethno-linguistic subgroups. CONCLUSION: This study revealed that one SNP rs1800794 of IL1A and one VNTR rs2234663 of IL1RN were associated with the increased risk to be infected by Trypanosoma brucei gambiense and develop sleeping sickness in southern Cameroon. The minor allele T and the genotype TT of SNP rs1800794 in IL1A as well as the genotype 1A3A of IL1RN rs2234663 VNTR seem to increase the risk of getting Trypanosoma brucei gambiense infections and develop sleeping sickness in southern Cameroon.

Macrophage migrating inhibitory factor expression is associated with Trypanosoma brucei gambiense infection and is controlled by trans-acting expression quantitative trait loci in the Guinean population.

Infection by Trypanosoma brucei gambiense is characterized by a wide array of clinical outcomes, ranging from asymptomatic to acute disease and even spontaneous cure. In this study, we investigated the association between macrophage migrating inhibitory factor (MIF), an important pro-inflammatory cytokine that plays a central role in both innate and acquired immunity, and disease outcome during T. b. gambiense infection. A comparative expression analysis of patients, individuals with latent infection and controls found that MIF had significantly higher expression in patients (n = 141; 1.25 ±â€¯0.07; p < .0001) and latent infections (n = 25; 1.23 ±â€¯0.13; p = .0005) relative to controls (n = 46; 0.94 ±â€¯0.11). Furthermore, expression decreased significantly after treatment (patients before treatment n = 33; 1.40 ±â€¯0.18 versus patients after treatment n = 33; 0.99 ±â€¯0.10, p = .0001). We conducted a genome wide eQTL analysis on 29 controls, 128 cases and 15 latently infected individuals for whom expression and genotype data were both available. Four loci, including one containing the chemokine CXCL13, were found to associate with MIF expression. Genes at these loci are candidate regulators of increased expression of MIF after infection. Our study is the first data demonstrating that MIF expression is elevated in T. b. gambiense-infected human hosts but does not appear to contribute to pathology.

FungiDB: An Integrated Bioinformatic Resource for Fungi and Oomycetes.

FungiDB (fungidb.org) is a free online resource for data mining and functional genomics analysis for fungal and oomycete species. FungiDB is part of the Eukaryotic Pathogen Genomics Database Resource (EuPathDB, eupathdb.org) platform that integrates genomic, transcriptomic, proteomic, and phenotypic datasets, and other types of data for pathogenic and nonpathogenic, free-living and parasitic organisms. FungiDB is one of the largest EuPathDB databases containing nearly 100 genomes obtained from GenBank, Aspergillus Genome Database (AspGD), The Broad Institute, Joint Genome Institute (JGI), Ensembl, and other sources. FungiDB offers a user-friendly web interface with embedded bioinformatics tools that support custom in silico experiments that leverage FungiDB-integrated data. In addition, a Galaxy-based workspace enables users to generate custom pipelines for large-scale data analysis (e.g., RNA-Seq, variant calling, etc.). This review provides an introduction to the FungiDB resources and focuses on available features, tools, and queries and how they can be used to mine data across a diverse range of integrated FungiDB datasets and records.

The Structure of a Conserved Telomeric Region Associated with Variant Antigen Loci in the Blood Parasite Trypanosoma congolense.

African trypanosomiasis is a vector-borne disease of humans and livestock caused by African trypanosomes (Trypanosoma spp.). Survival in the vertebrate bloodstream depends on antigenic variation of Variant Surface Glycoproteins (VSGs) coating the parasite surface. In T. brucei, a model for antigenic variation, monoallelic VSG expression originates from dedicated VSG expression sites (VES). Trypanosoma brucei VES have a conserved structure consisting of a telomeric VSG locus downstream of unique, repeat sequences, and an independent promoter. Additional protein-coding sequences, known as "Expression Site Associated Genes (ESAGs)", are also often present and are implicated in diverse, bloodstream-stage functions. Trypanosoma congolense is a related veterinary pathogen, also displaying VSG-mediated antigenic variation. A T. congolense VES has not been described, making it unclear if regulation of VSG expression is conserved between species. Here, we describe a conserved telomeric region associated with VSG loci from long-read DNA sequencing of two T. congolense strains, which consists of a distal repeat, conserved noncoding elements and other genes besides the VSG; although these are not orthologous to T. brucei ESAGs. Most conserved telomeric regions are associated with accessory minichromosomes, but the same structure may also be associated with megabase chromosomes. We propose that this region represents the T. congolense VES, and through comparison with T. brucei, we discuss the parallel evolution of antigenic switching mechanisms, and unique adaptation of the T. brucei VES for developmental regulation of bloodstream-stage genes. Hence, we provide a basis for understanding antigenic switching in T. congolense and the origins of the African trypanosome VES.

No evidence for association between APOL1 kidney disease risk alleles and Human African Trypanosomiasis in two Ugandan populations.

BACKGROUND: Human African trypanosomiasis (HAT) manifests as an acute form caused by Trypanosoma brucei rhodesiense (Tbr) and a chronic form caused by Trypanosoma brucei gambiense (Tbg). Previous studies have suggested a host genetic role in infection outcomes, particularly for APOL1. We have undertaken candidate gene association studies (CGAS) in a Ugandan Tbr and a Tbg HAT endemic area, to determine whether polymorphisms in IL10, IL8, IL4, HLAG, TNFA, TNX4LB, IL6, IFNG, MIF, APOL1, HLAA, IL1B, IL4R, IL12B, IL12R, HP, HPR, and CFH have a role in HAT. METHODOLOGY AND RESULTS: We included 238 and 202 participants from the Busoga Tbr and Northwest Uganda Tbg endemic areas respectively. Single Nucleotide Polymorphism (SNP) genotype data were analysed in the CGAS. The study was powered to find odds ratios > 2 but association testing of the SNPs with HAT yielded no positive associations i.e. none significant after correction for multiple testing. However there was strong evidence for no association with Tbr HAT and APOL1 G2 of the size previously reported in the Kabermaido district of Uganda. CONCLUSIONS/SIGNIFICANCE: A recent study in the Soroti and Kaberamaido focus in Central Uganda found that the APOL1 G2 allele was strongly associated with protection against Tbr HAT (odds ratio = 0.2, 95% CI: 0.07 to 0.48, p = 0.0001). However, in our study no effect of G2 on Tbr HAT was found, despite being well powered to find a similar sized effect (OR = 0.9281, 95% CI: 0.482 to 1.788, p = 0.8035). It is possible that the G2 allele is protective from Tbr in the Soroti/Kabermaido focus but not in the Iganga district of Busoga, which differ in ethnicity and infection history. Mechanisms underlying HAT infection outcome and virulence are complex and might differ between populations, and likely involve several host, parasite or even environmental factors.

Candidate genes-based investigation of susceptibility to Human African Trypanosomiasis in Côte d'Ivoire.

Human African Trypanosomiasis (HAT) or sleeping sickness is a Neglected Tropical Disease. Long regarded as an invariably fatal disease, there is increasing evidence that infection by T. b. gambiense can result in a wide range of clinical outcomes, including latent infections, which are long lasting infections with no parasites detectable by microscopy. The determinants of this clinical diversity are not well understood but could be due in part to parasite or host genetic diversity in multiple genes, or their interactions. A candidate gene association study was conducted in Côte d'Ivoire using a case-control design which included a total of 233 subjects (100 active HAT cases, 100 controls and 33 latent infections). All three possible pairwise comparisons between the three phenotypes were tested using 96 SNPs in16 candidate genes (IL1, IL4, IL4R, IL6, IL8, IL10, IL12, IL12R, TNFA, INFG, MIF, APOL1, HPR, CFH, HLA-A and HLA-G). Data from 77 SNPs passed quality control. There were suggestive associations at three loci in IL6 and TNFA in the comparison between active cases and controls, one SNP in each of APOL1, MIF and IL6 in the comparison between latent infections and active cases and seven SNP in IL4, HLA-G and TNFA between latent infections and controls. No associations remained significant after Bonferroni correction, but the Benjamini Hochberg false discovery rate test indicated that there were strong probabilities that at least some of the associations were genuine. The excess of associations with latent infections despite the small number of samples available suggests that these subjects form a distinct genetic cluster different from active HAT cases and controls, although no clustering by phenotype was observed by principle component analysis. This underlines the complexity of the interactions existing between host genetic polymorphisms and parasite diversity.

A polymorphism in the haptoglobin, haptoglobin related protein locus is associated with risk of human sleeping sickness within Cameroonian populations.

BACKGROUND: Human African Trypanosomiasis (HAT) is a neglected disease targeted for elimination as a public health problem by 2020. Elimination requires a better understanding of the epidemiology and clinical evolution of HAT. In addition to the classical clinical evolution of HAT, asymptomatic carriers and spontaneous cure have been reported in West Africa. A genetic component to human susceptibility to HAT has been suggested to explain these newly observed responses to infection. In order to test for genetic associations with infection response, genetic polymorphism in 17 genes were tested (APOL1, IL1B, IL4, IL4R, IL6, IL8, IL12B, IL12RB1, IL10, TNFA, INFG, MIF, HLA-G, HLA-A, HP, HPR and CFH). METHODOLOGY: A case-control study was performed on 180 blood samples collected from 56 cases and 124 controls from Cameroon. DNA was extracted from blood samples. After quality control, 25 samples (24 controls and 1 case) were eliminated. The genotyping undertaken on 155 individuals including 55 cases and 100 controls were investigated at 96 loci (88 SNPs and 8 indels) located on 17 genes. Associations between these loci and HAT were estimated via a case-control association test. RESULTS: Analyses of 64 SNPs and 4 indels out of 96 identified in the selected genes reveal that the minor allele (T) of rs8062041 in haptoglobin (HP) appeared to be protective against HAT (p = 0.0002395, OR 0.359 (CI95 [0.204-0.6319])); indicating higher frequency in cases compared to controls. This minor allele with adjusted p value of 0.0163 is associated with a lower risk (protective effect) of developing sleeping sickness. CONCLUSION: The haptoglobin related protein HPR and HP are tightly linked and both are duplicated in some people and may lead to higher activity. This increased production could be responsible of the protection associated with rs8062041 even though this SNP is within HP.

Leaf LIMS: A Flexible Laboratory Information Management System with a Synthetic Biology Focus.

This paper presents Leaf LIMS, a flexible laboratory information management system (LIMS) designed to address the complexity of synthetic biology workflows. At the project's inception there was a lack of a LIMS designed specifically to address synthetic biology processes, with most systems focused on either next generation sequencing or biobanks and clinical sample handling. Leaf LIMS implements integrated project, item, and laboratory stock tracking, offering complete sample and construct genealogy, materials and lot tracking, and modular assay data capture. Hence, it enables highly configurable task-based workflows and supports data capture from project inception to completion. As such, in addition to it supporting synthetic biology it is ideal for many laboratory environments with multiple projects and users. The system is deployed as a web application through Docker and is provided under a permissive MIT license. It is freely available for download at https://leaflims.github.io .

Candidate gene polymorphisms study between human African trypanosomiasis clinical phenotypes in Guinea.

BACKGROUND: Human African trypanosomiasis (HAT), a lethal disease induced by Trypanosoma brucei gambiense, has a range of clinical outcomes in its human host in West Africa: an acute form progressing rapidly to second stage, spontaneous self-cure and individuals able to regulate parasitaemia at very low levels, have all been reported from endemic foci. In order to test if this clinical diversity is influenced by host genetic determinants, the association between candidate gene polymorphisms and HAT outcome was investigated in populations from HAT active foci in Guinea. METHODOLOGY AND RESULTS: Samples were collected from 425 individuals; comprising of 232 HAT cases, 79 subjects with long lasting positive and specific serology but negative parasitology and 114 endemic controls. Genotypes of 28 SNPs in eight genes passed quality control and were used for an association analysis. IL6 rs1818879 allele A (p = 0.0001, OR = 0.39, CI95 = [0.24-0.63], BONF = 0.0034) was associated with a lower risk of progressing from latent infection to active disease. MIF rs36086171 allele G seemed to be associated with an increased risk (p = 0.0239, OR = 1.65, CI95 = [1.07-2.53], BONF = 0.6697) but did not remain significant after Bonferroni correction. Similarly MIF rs12483859 C allele seems be associated with latent infections (p = 0.0077, OR = 1.86, CI95 = [1.18-2.95], BONF = 0.2157). We confirmed earlier observations that APOL1 G2 allele (DEL) (p = 0.0011, OR = 2.70, CI95 = [1.49-4.91], BONF = 0.0301) is associated with a higher risk and APOL1 G1 polymorphism (p = 0.0005, OR = 0.45, CI95 = [0.29-0.70], BONF = 0.0129) with a lower risk of developing HAT. No associations were found with other candidate genes. CONCLUSION: Our data show that host genes are involved in modulating Trypanosoma brucei gambiense infection outcome in infected individuals from Guinea with IL6 rs1818879 being associated with a lower risk of progressing to active HAT. These results enhance our understanding of host-parasite interactions and, ultimately, may lead to the development of new control tools.

SMRT Gate: A method for validation of synthetic constructs on Pacific Biosciences sequencing platforms.

Current DNA assembly methods are prone to sequence errors, requiring rigorous quality control (QC) to identify incorrect assemblies or synthesized constructs. Such errors can lead to misinterpretation of phenotypes. Because of this intrinsic problem, routine QC analysis is generally performed on three or more clones using a combination of restriction endonuclease assays, colony PCR, and Sanger sequencing. However, as new automation methods emerge that enable high-throughput assembly, QC using these techniques has become a major bottleneck. Here, we describe a quick and affordable methodology for the QC of synthetic constructs. Our method involves a one-pot digestion-ligation DNA assembly reaction, based on the Golden Gate assembly methodology, that is coupled with Pacific Biosciences' Single Molecule, Real-Time (PacBio SMRT) sequencing technology.

EuPathDB: the eukaryotic pathogen genomics database resource.

The Eukaryotic Pathogen Genomics Database Resource (EuPathDB, http://eupathdb.org) is a collection of databases covering 170+ eukaryotic pathogens (protists & fungi), along with relevant free-living and non-pathogenic species, and select pathogen hosts. To facilitate the discovery of meaningful biological relationships, the databases couple preconfigured searches with visualization and analysis tools for comprehensive data mining via intuitive graphical interfaces and APIs. All data are analyzed with the same workflows, including creation of gene orthology profiles, so data are easily compared across data sets, data types and organisms. EuPathDB is updated with numerous new analysis tools, features, data sets and data types. New tools include GO, metabolic pathway and word enrichment analyses plus an online workspace for analysis of personal, non-public, large-scale data. Expanded data content is mostly genomic and functional genomic data while new data types include protein microarray, metabolic pathways, compounds, quantitative proteomics, copy number variation, and polysomal transcriptomics. New features include consistent categorization of searches, data sets and genome browser tracks; redesigned gene pages; effective integration of alternative transcripts; and a EuPathDB Galaxy instance for private analyses of a user's data. Forthcoming upgrades include user workspaces for private integration of data with existing EuPathDB data and improved integration and presentation of host-pathogen interactions.