James Wasmuth

The University of Calgary · Department of Ecosystem and Public Health

Background Structural variant (SV) calling from DNA sequencing data has been challenging due to several factors, including the ambiguity of short-read alignments, multiple complex SVs in the same genomic region, and the lack of “truth” datasets for benchmarking. Additionally, caller choice, parameter settings, and alignment method are known to affe...

Genome assemblers are a critical component of genome science, but the choice of assembly software and protocols can be daunting. Here, we investigate genome assembly variation and its implications for gene discovery across three nematode species--Caenorhabditis bovis, Haemonchus contortus, and Heligmosomoides bakeri--highlighting the critical inter...

Single-cell atlases aim to collect the gene expression information for every cell type in an organism but can be challenging to perform in non-model organisms. To try to circumvent the problem of having no verified cell type markers in the parasitic nematode Heligmosomoides bakeri to use for an atlas, we attempted to use orthologs of verified marke...

Background The Myxozoa is a group of at least 2,400 endoparasites within the phylum Cnidaria. All myxozoans have greatly reduced in size and morphology compared to free-living members of the phylum. They are best known for causing disease in economically important fish across the world; for example, Myxobolus cerebralis causes Whirling Disease, whi...

HpARI is an immunomodulatory protein secreted by the intestinal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains three HpARI family members and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing and HpARI3 a...

Pathogens have evolved sophisticated strategies to manipulate host signaling pathways, including the phenomenon of molecular mimicry, where pathogen-derived biomolecules imitate host biomolecules. In this study, we resurrected, updated, and optimized a sequence-based bioinformatics pipeline to identify potential molecular mimicry candidates between...

HpARI is an immunomodulatory protein secreted by the intesYnal nematode Heligmosomoides polygyrus bakeri, which binds and blocks IL-33. Here, we find that the H. polygyrus bakeri genome contains 3 HpARI family members, and that these have different effects on IL-33-dependent responses in vitro and in vivo, with HpARI1+2 suppressing, and HpARI3 ampl...

Background Heligmosomoides bakeri (often mistaken for Heligmosomoides polygyrus ) is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between n...

Background Structural variation (SV) calling from DNA sequencing data has been challenging due to several factors, such as the ambiguity of short-read alignments, multiple complex SVs in the same genomic region, and the lack of “truth” datasets for benchmarking. Additionally, caller choice, parameter settings, and alignment method are known to affe...

The accurate characterization of structural variation is crucial for our understanding of how large chromosomal alterations affect phenotypic differences and contribute to genome evolution. Whole-genome sequencing is a popular approach for identifying structural variants, but the accuracy of popular tools remains unclear due to the limitations of e...

Introduction Intestinal roundworms cause chronic debilitating disease in animals, including humans. Traditional experimental models of these types of infection use a large single-dose infection. However, in natural settings, hosts are exposed to parasites on a regular basis and when mice are exposed to frequent, smaller doses of Heligmosomoides pol...

Heligmosomoides polygyrus is a promising model for parasitic nematodes with the key advantage of being amenable to study and manipulation within a controlled laboratory environment. While draft genome sequences are available for this worm, which allow for comparative genomic analyses between nematodes, there is a notable lack of information on its...

Genome-wide methods offer a powerful approach to detect signatures of drug selection. However, limited availability of suitable reference genomes and the difficulty of obtaining field populations with well-defined, distinct drug treatment histories mean there is little information on the signatures of selection in parasitic nematodes and on how bes...

Genome-wide methods offer a powerful approach to detect signatures of drug selection in parasite populations in the field. However, their application to parasitic nematodes has been limited because of both a lack of suitable reference genomes and the difficulty of obtaining field populations with sufficiently well-defined drug selection histories....

Cyclospora cayetanensis is an emerging foodborne parasite that causes cyclosporiasis, an enteric disease of humans. Domestically acquired outbreaks have been reported in Canada every spring or summer since 2013. To date, investigations into the potential sources of infection have relied solely on epidemiological data. To supplement the epidemiologi...

The apicomplexan parasite Cyclospora cayetanensis causes foodborne gastrointestinal disease in humans. Here, we report the first hybrid assembly for C. cayetanensis, which uses both Illumina MiSeq and Oxford Nanopore Technologies MinION platforms to generate genomic sequence data. The final genome assembly consists of 44,586,677 bases represented i...

The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with...

The intestinal roundworm Heligmosomoides bakeri causes chronic infection in susceptible (C57Bl/6) mice; however, repeat (trickle) infection confers immunity and facilitates worm clearance. We previously showed that this acquired immunity is associated with a strong Th2 response, notably the enhanced production of intestinal granulomas. Here we demo...

Background: Currently available short read genome assemblies of the tetraploid protozoan parasite Giardia intestinalis are highly fragmented, highlighting the need for improved genome assemblies at a reasonable cost. Long nanopore reads are well suited to resolve repetitive genomic regions resulting in better quality assemblies of eukaryotic genom...

Background Genomes of the parasite Giardia duodenalis are relatively small for eukaryotic genomes, yet there are only six publicly available. Difficulties in assembling the tetraploid G. duodenalis genome from short read sequencing data likely contribute to this lack of genomic information. We sequenced three isolates of G. duodenalis (AWB, BGS, an...

After transitioning to a new environment, species often exhibit rapid phenotypic innovation. One of the fastest mechanisms for this is duplication followed by specialization of existing genes. When this happens to a member of a gene family, it tends to leave a detectable phylogenetic signature of lineage-specific expansions and contractions. These...

Supporting information. Table S1: The nematode collagens identified in this work from species in the genus Caenorhabditis. Article S1: The MIPhy model of gene family evolution. Figure S1: Example species and gene trees.

Data from collagen analysis. The following files are contained in the tarball: all_caen.col2.fas - collagen sequences from seven species of Caenorhabditis in fasta format. MIPhy_scores.csv - comma separated file generated by MIPhy. (1) sequence identifier; (2) species identifier; (3) MIPhy cluster identifier; (4) Instability score. withPheno.txt -...

We present DETECT v2-an enzyme annotation tool which considers the effect of sequence diversity when assigning enzymatic function (as an Enzyme Commission number) to a protein sequence. In addition to capturing more enzyme classes than the previous version, we now provide EC-specific cutoffs that greatly increase precision and recall of assignments...

After transitioning to a new environment, species often exhibit rapid phenotypic innovation. One of the fastest mechanisms for this is duplication followed by specialization of existing genes, which leaves a phylogenetic signature of lineage-specific expansions and contractions. These can be identified by analyzing the gene family across several sp...

After transitioning to a new environment, species often exhibit rapid phenotypic innovation. One of the fastest mechanisms for this is duplication followed by specialization of existing genes, which leaves a phylogenetic signature of lineage-specific expansions and contractions. These can be identified by analyzing the gene family across several sp...

Background Signalling pathways underlie development, behaviour and pathology. To understand patterns in the evolution of signalling pathways, we undertook a comprehensive investigation of the pathways that control the switch between growth and developmentally quiescent dauer in 24 species of nematodes spanning the phylum. Results Our analysis of 4...

Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, ep...

File includes all relevant data used to generate the Figs 2, 3, 4, 5 and 6. The data included is in the form of number of reads assigned to each species. (XLSX)

Schematic representation of the preparation of the Illumina sequencing Library. In the initial PCR amplification, overhanging primers are used to amplify the ITS-2 rDNA region to generate a fragment that includes the ITS-2 amplicon as well as the Rd1 and Rd2 specific primer regions. The Rd1 and Rd2 regions provide the target sites for the primers u...

PCR primer sequences for library preparation. S1A) Primer sequences for ITS-2 Amplification. NC1/NC2 Primer sequence bolded, N’s are underlined. S1B) Forward Indexed Primers. Index sequence bolded S1C) Reverse Indexed Primers. Index sequence bolded. (EPS)

ITS-2 consensus sequences and fixed SNP differences between parasite species. S3A) ITS-2 sequences were generated from 15 (14 for Cooperia oncohphora) rDNA ITS-2 sequences obtained from individual larvae for each of 8 species as well as all ITS-2 sequences available from Genbank at time of publication. Sequences were analyzed using Geneious version...

Inter and Intra species variation of ITS-2 Sequence Identity. A) Consensus sequences were generated from 15 (14 for Cooperia oncohphora) rDNA ITS-2 sequences obtained from individual larvae for each of 8 species. Sequences were analyzed using Geneious version 7.1.5, and trimmed to remove the 5.8S and 28S regions leaving only the ITS-2 locus. Sequen...

The impact of the microbiota on the immune status of its host is a source of intense research and publicity. In comparison, the effect of arthropod microbiota on vector-borne infectious diseases has received little attention. A better understanding of the vector microbiota in relation to mammalian host immune responses is vital, as it can lead to s...

Abstract The hookworms Uncinaria rauschi Olsen, 1968 and Uncinaria yukonensis (Wolfgang, 1956) were formally described from grizzly (Ursus arctos horribilis) and black bears (Ursus americanus) of North America. We analyzed the intestinal tracts of 4 grizzly and 9 black bears from Alberta and British Columbia, Canada, and isolated Uncinaria specimen...

Kablammo is a web-based application that produces interactive, vector-based visualizations of sequence alignments generated by BLAST. These visualizations can illustrate many features, including shared protein domains, chromosome structural modifications, and genome misassembly. Availability and implementation: Kablammo can be used at http://kablam...

Motivation: Gene models from draft genome assemblies of metazoan species are often incorrect, missing exons or entire genes, particularly for large gene families. Consequently, labour-intensive manual curation is often necessary. We present Figmop (Finding Genes using Motif Patterns) to help with the manual curation of gene families in draft genom...

Genomes and genomics are now part of the popular imagination and culture. Understanding what these massively long strings of As, Gs, Cs, and Ts actually mean is a challenge that has been taken up by many working on parasites. Our understanding of parasite biology and future treatment strategies has been significantly improved because of these genom...

Increasingly, metabolic potential is proving to be a critical determinant governing a pathogen's virulence as well as its capacity to expand its host range. To understand the potential contribution of metabolism to strain-specific infectivity differences, we present a constraint-based metabolic model of the opportunistic parasite, Toxoplasma gondii...

The small ruminant parasite Haemonchus contortus is the most widely used parasitic nematode in drug discovery, vaccine development and anthelmintic resistance research. Its remarkable propensity to develop resistance threatens the viability of the sheep industry in many regions of the world and provides a cautionary example of the effect of mass dr...

The health and economic importance of parasitic nematodes cannot be overstated. Moreover, they offer a complex and diverse array of life strategies, raising a multitude of evolutionary questions. Researchers are applying population genetics to parasitic nematodes in order to disentangle some aspects of their life strategies, improve our knowledge a...

Tapeworms (Cestoda) cause neglected diseases that can be fatal and are difficult to treat, owing to inefficient drugs. Here we present an analysis of tapeworm genome sequences using the human-infective species Echinococcus multilocularis, E. granulosus, Taenia solium and the laboratory model Hymenolepis microstoma as examples. The 115- to 141-megab...

Parasitic nematode β-tubulin genes are of particular interest because they are the targets of benzimidazole drugs. However, in spite of this, the full β-tubulin gene family has not been characterized for any parasitic nematode to date. Haemonchus contortus is the parasite species for which we understand benzimidazole resistance the best and its clo...

: Malaria and HIV-1 adversely interact, with HIV-positive individuals suffering higher parasite burdens and worse clinical outcomes. However, the mechanisms underlying these disease interactions are unclear. We hypothesized that HIV coinfection impairs the innate immune response to malaria, and that combination antiretroviral therapy (cART) may res...

Background: The Tim-3 receptor has been implicated as a negative regulator of adaptive immune responses and has been linked to T-cell dysfunction in chronic viral infections, such as HIV. Blocking Tim-3 has been proposed as a potential therapeutic intervention in HIV infection. However, a more detailed characterization of Tim-3 expression in the p...

Patients with platelet α or dense δ-granule defects have bleeding problems. Although several proteins are known to be required for δ-granule development, less is known about α-granule biogenesis. Our previous work showed that the BEACH protein NBEAL2 and the Sec1/Munc18 protein VPS33B are required for α-granule biogenesis. Using a yeast two-hybrid...

[This corrects the article on p. e1897 in vol. 6.].

BLAST bit score distribution of Trematode and Tricladid matches to E. granulosus sequences. Graphs indicate the number of E. granulosus matches to three different datasets: i) all Trematode sequences (74,794 sequences); ii) all Tricladid sequences (22,327 sequences); and iii) 22,327 randomly selected Trematode sequences (100 samples – standard devi...

Summary of E. granulosus EST clusters. (XLS)

Manually assembled contigs from EgBRep containing ESTs. (XLS)

Comparison of E. granulosus and related cestode tetraspanins. Full-length EgTSPs identified in our dataset were aligned with highly similar proteins from E. multilocularis (Em-TSP1, 5 and 6) and T. solium (Ts-T24, the ortholog of Em-TSP5; [120]). Fully conserved residues are marked with (*), those replaced with amino acids of strongly similar prope...

Unlabelled: The Toxoplasma gondii SRS gene superfamily is structurally related to SRS29B (formerly SAG1), a surface adhesin that binds host cells and stimulates host immunity. Comparative genomic analyses of three Toxoplasma strains identified 182 SRS genes distributed across 14 chromosomes at 57 genomic loci. Eight distinct SRS subfamilies were r...

Structure-guided alignment of each domain subfamily. Download Figure S1, PDF file, 0.3 MB.

Single-nucleotide polymorphisms in 69 core SRS genes and ω values.

Evaluation of EST and microarray data confirming transcript expression for 95 SRS genes.

Primer and probe sets used for qRT-PCR.

Detailed Materials and Methods. Download File S1, DOCX file, 0.1 MB.

The cestode Echinococcus granulosus - the agent of cystic echinococcosis, a zoonosis affecting humans and domestic animals worldwide - is an excellent model for the study of host-parasite cross-talk that interfaces with two mammalian hosts. To develop the molecular analysis of these interactions, we carried out an EST survey of E. granulosus larval...

Distribution of SRS domain architecture subfamilies. Alignments of each chromosome are shown with N. caninum above T. gondii. Pseudogenes are not shown in this figure. ‘VEG’ identifies loci where genes differ between Me49 and VEG strains of T. gondii. N.b. Note an expansion of family 3 in T. gondii Me49 at the start of chromosome VI. Family 3 has o...

De novo identification of non-coding RNAs. The methodology used to identify ncRNAs is shown in A. B shows the overlap in those RNA candidates predicted to have structure by three different methods RNAz [4], Pfold [5] and QRNA [6]. Further details are given in the SOM text. HSP = High-scoring Segment Pair. (EPS)

Evidence for overlapping UTR sequences in convergently transcribed genes in both N. caninum and T. gondii. We identified at least one convincing example of overlapping UTRs although we believe this is probably a rare occurrence in T. gondii and N. caninum. We show the example of NCLIV_009490 and NCLIV_009500 in N. caninum and their syntenic ortholo...

Identity and orthologous relationships for apical complex genes and AP2 transcription factors. T. gondii genes whose products are known to localize to apical complex organelles and their homologues (and AP2 transcription factors) are listed here. Where they exist, N. caninum othologues are identified and it is noted whether they occur in synteny or...

Putative early bradyzoite differentiation in N. caninum at day six of culture. (DOC)

Alignment of members of Lysine-Arginine rich Unidentified Function (KRUF). See Figure S legend for details. TgKRUF1 - TGME49_092400, TgKRUF2 - TGME49_010590, TgKRUF3 - TGME49_092390, TgKRUF4 - TGME49_051170, TgKRUF5 - TGME49_051060, TgKRUF6 - TGME49_095940, TgKRUF7 - TGME49_052180, NcKRUF8 - NCLIV_002020, NcKRUF9 - NCLIV_043740, NcKRUF10 - NCLIV_00...

Overrepresented GO terms in genes differentially expressed between days three and six of N. caninum tachyzoite culture. The term “DNA binding” largely refers to ApiAP2 transcription factor genes. “Protein amino acid phosphorylation” includes several rhoptry genes. (EPS)

Species-specific genes with functional information and additional evidence. RPKM values are means between replicates and are cut off below 6, as this was determined to be the minimum significant expression level. For Neospora we show RPKM values for days three, four and six of the tachyzoite stage and for Toxoplasma days three and four. T. gondii t...

RNAseq mapping statistics for N. caninum Liverpool tachyzoites. TZ = tachyzoite. Sequencing was performed using an Illumina GAII as described in methods. Raw reads for these runs are available from the European Nucleotide Archive (ENA; http://www.ebi.ac.uk/ena/) under the run id listed. In library names A and B refer to biological replicates for a...

Evidence of gene conversion at the SRS19 locus but not at the SRS29 locus. Genes at the SRS19 locus cluster based on species rather than as orthologous pairs suggesting they have been subject to gene conversion (A). N. caninum genes are highlighted in purple. Genes from three representative strains of T. gondii are shown (ME49, GT1, VEG). The SRS37...

Alignment and predicted structure of a novel ncRNA expanded in N. caninum. Alignment of predicted novel ncRNA group 56 (A). The secondary structure was predicted by the program RNAalifold from the Vienna Package [7] and shown below as less than’ and ‘greater than’ symbols. Residues able to form base pairs according to the predicted structure are hi...

RNAseq mapping statistics for T. gondii VEG tachyzoites. T. gondii RNAseq experiments were performed using the VEG strains due to their amenity in the lab, while the Me49 genome was used for mapping due to its superior assembly and annotation. Example day three and four RNAseq runs were mapped to the T. gondii VEG strain genome sequence to determin...

Identifying Non-coding RNA genes. (DOC)

Alignment of Toxoplasma-Specfic Family (TSF). Only sequences belonging to the Me49 strain of T. gondii are shown. Alignments here and in Supplementary Figure 2 were performed using Muscle [1] and displayed using Jalview [2]. Colouring is in Clustal format. TgTSF1 - TGME49_121170, TgTSF2 -TGME49_000700, TgTSF3 - TGME49_107260, TgTSF4 - TGME49_000130...

Identifying a threshold for calling gene expression. We observed noisy mRNA-seq read mapping e.g. to intergenic and intronic sequence. We could not therefore rely on only a single read to call a gene as expressed. In order to determine a threshold expression level above which we could confidently call a gene as expressed we examined levels of expre...

Frequencies of different SAG domain architectures. SRS genes comprise one or more copies of the SAG domain. The SAG domain superfamily has been classified into eight subfamilies Fam1 to Fam8 (Wasmuth et al., submitted). The domain architectures of SRS genes and their frequency of occurrence in the Neospora and Toxoplasma genomes are described below...

N. caninum isolates for which the ROP18 region was amplified and sequenced. (DOCX)

Varieties of tRNA genes found in T. gondii Me40 and N. caninum Nc-Liv genomes. (DOCX)

References for Tables S1, S2, S3, S4, S5, S6. (DOCX)

Toxoplasma gondii is a zoonotic protozoan parasite which infects nearly one third of the human population and is found in an extraordinary range of vertebrate hosts. Its epidemiology depends heavily on horizontal transmission, especially between rodents and its definitive host, the cat. Neospora caninum is a recently discovered close relative of To...

Routine fecal examination revealed novel coccidian oocysts in asymptomatic California sea lions (Zalophus californianus) in a rehabilitation facility. Coccidian oocysts were observed in fecal samples collected from 15 of 410 California sea lions admitted to The Marine Mammal Center between April 2007 and October 2009. Phylogenetic analysis using th...

Establishing the risk of human infection is one of the goals of public health. For bacterial pathogens, the virulence and zoonotic potential can often be related to their host source. Escherichia coli bacteria are common contaminants of water associated with human recreation and consumption, and many strains are pathogenic. In this study, we analyz...

Nematode parasites are of major importance in human health and agriculture, and free-living species deliver essential ecosystem services. The genomics revolution has resulted in the production of many datasets of expressed sequence tags (ESTs) from a phylogenetically wide range of nematode species, but these are not easily compared. NEMBASE4 presen...

1444 Human platelets contain α-granules, dense (δ-) granules and lysosomes that release their contents upon platelet activation. Platelet granule release is important for hemostasis, since patients with inherited granule defects have bleeding problems. α-granules are absent in the gray platelet and ARC syndromes, while deficient δ-granules are obse...

A major challenge in genomics is the accurate annotation of component genes. Enzymes are typically predicted using homology-based search methods, where the membership of a protein to an enzyme family is based on single-sequence comparisons. As such, these methods are often error-prone and lack useful measures of reliability for the prediction. Here...

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to...

Toxoplasma gondii, the etiological agent of toxoplasmosis, utilizes stage-specific expression of antigenically distinct glycosylphosphatidylinositol-tethered surface coat proteins to promote and establish chronic infection. Of the three infective stages of T. gondii, sporozoites are encapsulated in highly infectious oocysts that have been linked to...

Supplementary figures S1–5, Supplementary tables SI–II

InParanoid derived orthology relationships for Nedd4 substrates across 111 eukaryotes. Each tile in the heatmap depicts the predicted orthology relationship between the human Nedd4 substrate with genes from other organisms.

Ubiquitylation substrates and binding substrates (hits) for rat Nedd4-1 (rNedd4-1), human Nedd4-1 (hNedd4-1) and human Nedd4-2 (hNedd4-2) identified in the proteome microarray screens.

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2-4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global su...

The Apicomplexa are a group of phylogenetically related parasitic protists that include Plasmodium, Cryptosporidium, and Toxoplasma. Together they are a major global burden on human health and economics. To meet this challenge, several international consortia have generated vast amounts of sequence data for many of these parasites. Here, we exploit...

The genomes of an increasing number of species are being investigated through the generation of expressed sequence tags (ESTs). However, ESTs are prone to sequencing errors and typically define incomplete transcripts, making downstream annotation difficult. Annotation would be greatly improved with robust polypeptide translations. Many current solu...

Translation of the abstract into German by Ralf Schmid. Übersetzung der Zusammenfassung ins Deutsche von Ralf Schmid. (0.03 MB DOC)

Metabolism table: loss/gain (0.03 MB XLS)

Species distribution of novel domains (0.33 MB PDF)

Novel NemDom3 domains also identified in plants (includes protein identifiers) (0.45 MB PDF)

Translation of the abstract into French by Douglas Finney. Traduction du résumé en français par Douglas Finney. (0.03 MB DOC)