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Dillman, Adler, Rogers, Alicia, Adams, Byron, Williams, Brian, Macchietto, Marissa, Antoshechkin, Igor, Lewis, Edwin, Finlinson, Camille, Lu, Xiaojun, Goodrich-Blair, Heidi, Mortazavi, Ali, Sternberg, Paul, Goodwin, Zane, Stock, Patricia
[
International Worm Meeting,
2013]
Numerous nematode genera are major parasites of plants, animals, and humans, despite sharing a conserved body plan. Steinernema comprise over 70 characterized species that are lethal parasites of insects with differing foraging strategies and host ranges. We have sequenced the genomes and transcriptomes of five key members of Steinernema (S. carpocapsae, S. scapterisci, S. monticolum, S. glaseri, and S. feltiae) for comparative analysis. We find 20 Mb of conserved sequence, which represents about 23% of the S. carpocapsae assembly. This includes 127,282 non-coding elements accounting for about 5 Mb. We explore genomic differences likely to be involved in insect parasitism. We find gene family evolution of proteases, protease inhibitors, proteolytic cascade proteins, and GPCRs, many of which correlate with known differences in host range and specificity. Steinernema RNA-seq data allows for powerful comparisons to Caenorhabditis gene expression at defined stages, which show surprising plasticity of timing across one-to-one orthologous genes when compared to C. elegans. Our analysis of the conserved non-coding regions reveals that a limited number of motifs are associated with conservation of stage-specific ortholog expression, which suggests that key underlying gene regulatory relationships that control development are similar in the two genera.
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Schwarz, Erich M., Schaeffer, Lorian, Williams, Brian, Sternberg, Paul, Wold, Barbara, Mortazavi, Ali
[
International Worm Meeting,
2009]
The de novo sequencing of nematode genomes has been an arduous process that involves large-scale projects working over multi-year time scales to sequence and annotate genomes. The recent advent of ultra-high throughput sequencers that are moving towards the $1,000 human genome foreshadow the coming of the "$50 worm genome" for sequencing reagents, which will afford a much larger scale whole-genome survey of the nematode phylum. In order to develop tools to analyze and annotate nematode genomes of interest using ultra-high-throughput technology, we have sequenced the genome and the transcriptome of Caenorhabditis sp. 3 PS1010 using 2x75 bp reads produced on an Illumina GAII. We have been able to compare our genomic DNA and cDNA sequence data to 417 kb of high-quality, annotated contigs built using traditional sanger sequencing of PS1010 fosmids. We assembled 49 million paired reads into 65.5 Mb using the Velvet short read assembler with an N50 of 1.1 kb which achieved 95% coverage of our PS1010 contigs, for which the gene-prediction program AUGUSTUS predicted ~30,000 protein-coding genes or segments of genes. We also sequenced a pool of mixed-stage, polyA-selected RNA with over 26 million mappable reads (including 3.7 million splice-crossing reads), and found that we observed reliable signal of at least 1 or more Reads Per Kb per Million (RPKM) over 75% of the 108,000 AUGUSTUS-predicted exons; this includes developmental control genes expected to be expressed at low levels, such as
lin-3 and
lin-11. By taking advantage of the paired-end RNA-seq reads, we were able to further improve our assembly using RNA-reads spanning contigs and thus increase our N50 to 1.6 kb. The combination of ultra-high throughput sequencing of genomic DNA and of the transcriptome along with their complementary assembly provides a straightforward path for the further analysis of key species in the nematode phylum.
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[
International Worm Meeting,
2017]
Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most well-studied entomopathogens, due to its broad lethality to diverse insect species, and to its effective commercial use as a biological control agent for insect pests. For this reason, it has become an important genetic model for studying parasitism, pathogenesis, and symbiosis. We used a newly published hybrid assembly pipeline to assemble the best genome of S. carpocapsae to date, comprising 86,259,276 bp in 86 scaffolds, with an N50 of 4.03 Mb, from a combination of 75X coverage Pacbio and 130X coverage Illumina reads. We found that 90% of the genome is represented by the 22 largest scaffolds. RNA-seq data from 17 developmental stages spanning the embryo to adult stages were used to help predict 22,295 gene models, a major reduction in the number of genes from the previously published assembly by Dillman et al. 2015, which has 28,313 genes, and an increase in the number of genes relative to a high contiguity S. carpocapsae Breton strain assembly (N50=1.24), which has 16,333 genes. Using this new genome, we infer the potential chromosomal origins of our scaffolds by comparing them to C. elegans using shared one-to-one orthologs and find that many of the largest scaffolds correspond primarily to single chromosomes in C. elegans. We also investigate a potential large 1.2 Mb duplication in the genome, and delve into gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations will provide a good foundation for new comparative genomic and gene expression studies.
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[
International Worm Meeting,
2013]
Hox genes are evolutionarily conserved transcription factors that regulate the expression of other developmental genes. They are organized into clusters, with the order of genes in each cluster paralleling their expression along the anterior-posterior axis. In the free-living nematode Caenorhabditis elegans, the Hox genes are much more dispersed along the chromosome, and the anterior Hox genes,
ceh-13 and
lin-39, are reversed, but little is known about the Hox genes in other nematode taxa. We are interested in the Hox gene cluster architecture of insect parasitic nematodes from the genus Steinernema (S. carpocapsae, S. scapterisci, S. feltiae, S. glaseri, and S. monticolum), for which we have assembled genomes and stage-specific transcriptomes. More specifically, we are interested in exploring the presence, order, and dispersal of the Hox genes in these newly sequenced nematodes, to identify the level of conservation within this genus and their conservation with C. elegans. We also investigated the extent of non-coding conservation around Hox genes, looking for candidate regulatory regions. Surprisingly, the Hox gene cluster among steinernematids is very different from what is known in C. elegans. For example, we have found approximately 10 genes between the steinernematid Hox genes
ceh-13 and
lin-39, whereas the region between the orthologous
ceh-13 and
lin-39 Hox genes in C. elegans is a gene desert. Interestingly, many of the intervening genes in the steinernematids are conserved and expressed in C. elegans, but are located nowhere near the C. elegans Hox cluster. We explored this in another nematode genome that we have assembled, Panagrellus redivivus, where we find only two intervening genes present between
ceh-13 and
lin-39 Hox genes. These findings suggest that the organization of the Hox cluster in nematodes has structural plasticity and varies across the phylum.
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[
International Worm Meeting,
2015]
Cells express distinct sets of genes in a precise spatio-temporal manner during embryonic development. There is a wealth of information about embryonic development in C. elegans, but much less is known about embryonic development at the molecular level in nematodes from other taxa. We are interested in insect pathogenic nematodes from the genus Steinernema as models of parasitism and symbiosis as well as satellite model for evolution in comparison to C. elegans. We determined the timing of embryonic developmental stages in three Steinernema species (S. carpocapsae, S. feltiae, and S. glaseri) for which we have assembled genomes. We found that the timing between embryonic developmental stages in Steinernema is longer than in C. elegans, and that the timing also varies among Steinernema species. We then sequenced the transcriptomes of single embryos of each species during embryonic development at specific stages (2-cell, 4-cell, 8-cell, comma, and 2-fold) for comparative analysis. Approximately two-thirds of all genes in S. carpocapsae and S. feltiae were expressed (> 1 FPKM) throughout embryogenesis. Single embryo transcriptomes correlated within early and late developmental stage embryos, but not between early and late embryos. An analysis of gene expression dynamics in S. feltiae revealed 5,513 genes with significant temporal dynamics in 8 distinct clusters, which have specific gene ontology enrichments. In the future, we will compare the temporal expression of Steinernema and C. elegans orthologs to determine their degree of temporal conservation during development between these distantly related species. .
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[
International Worm Meeting,
2015]
Living organisms rely on genes to manage all aspects of their lives. Although all of the cells in an organism possess the exact same genetic code, they can specialize to form different tissues and organs by selectively expressing particular sets of genes at particular times. We designed a modular course to introduce high school students to these major genetic concepts and the sequencing technologies that are now revolutionizing the field of genomics. The course focuses on nematodes of the genus Steinernema, insect parasites with broad scientific and commercial applications. Each module achieves specific teaching goals, and they can be used alone or in combination to meet the particular needs of individual instructors. Using this approach, we were able to guide students through all the stages of a modern genomics experiment: culturing the organism of study, isolating RNA from different stage of Steinernema life cycle, sequencing the libraries, and then analyzing the data using open-source computational tools. Students reacted positively to their hands-on experience performing the RNA-seq assay and analyzing the data they generated, but they especially enjoyed being given the opportunity to design their own projects based on the nematodes. Overall, we have demonstrated that sequencing assays and genomics can be taught to high school students, and that this course format makes this field more accessible to teachers and students at the secondary and post-secondary levels.
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[
International Worm Meeting,
2017]
We are interested in using entomopathogenic nematodes (EPNs) from the family Steinernematidae, which are nematodes that parasitize and efficiently kill insects and are used as satellite model organism, to study the conservation of endoderm development in nematodes. The Steinernema carpocapsae genome lacks the GATA transcription factors END-1 and END-3, which control endoderm development in the E-cell of the 8-cell stage in C. elegans while their downstream target genes are conserved and expressed abundantly during endoderm development. Therefore, there must be an alternative set of early-expressed Transcription Factors(TFs) that determine endoderm cell fate in S. carpocapsae. We are isolating single-cells from S. carpocapsae and have sequenced individual single-cells from early stages of S. carpocapsae to identify early zygotic TFs that could be cell lineage specific. The embryonic localization of these TFs will be verified using single molecule fluorescent in situ hybridization(smFISH). We will compare our results in S. carpocapsae to the matching single-cell data from C. elegans E-cells to perform the first comparison of gene expression at the single-cell level among homologous cells across distant nematode species with a focus on regulatory genes controlling early endoderm development.
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[
International Worm Meeting,
2011]
Nematodes form one of the largest invertebrate phyla with an estimated one million species occupying every conceivable niche. Besides the free-living model organism Caenorhabditis elegans, studied nematodes include parasites of plants, insects, livestock, and humans. Parasitic species often display great specificity to their hosts. The genus Steinernema comprises over 55 well-characterized species that are lethal parasites of insects with differing host ranges. We have sequenced and assembled the genome and staged transcriptomes of five whole genomes spanning the Steinernema genus (S. carpocapsae, S. scapterisci, S. monticolum, S. glaseri, and S. feltiae) using the Illumina platform. Steinernematid genomes prove amenable to Illumina sequencing due to their size (~95 Mb) and high G+C content (~45%). The combination of multiple closely related genomes in a non-Caenorhabidtis clade and accompanying deeply sequenced transcriptomes allows for powerful comparisons to other genera such as Caenorhabditis. In particular, comparisons in expression at defined stages shows significant plasticity of timing across one-to-one orthologous genes in the 5 genomes plus C. elegans. We explore the nematode contribution to mutualism using SSH library comparisons between S. carpocapsae grown on its symbiont, Xenhorhabdus nematophila, and S. carpocapsae grown on an X. nematophila colonization defective mutant. We identify nematode genes that are likely involved in colonization and explore their conservation and expression within the genus. We further examine the utility of these five genomes by orthology analysis within Nematoda, assessing the conservation of biological pathways, analyzing regulatory regions, and identify candidate genes involved in niche partitioning, host range, and mutualism within Steinernema.
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Macchietto, Marissa, Murad, Rabi, Maya Rodriguez, Isaryhia, Serra, Lorrayne, Macias-Munoz, Aide, Mortazavi, Ali, Rodriguez, Bryan, Joan McGill, Cassandra
[
International Worm Meeting,
2019]
Entomopathogenic nematodes from the genus Steinernema are lethal insect parasites that quickly kill their insect hosts with the help of their symbiotic bacteria. Steinernema carpocapsae is one of the most studied entomopathogens due to its broad lethality to diverse insect species and its effective commercial use as a biological control agent for insect pests, as well as a genetic model for studying parasitism, pathogenesis, and symbiosis. In this study, we used long-reads from the Pacific Biosciences platform and BioNano Genomics Irys system to assemble the best genome of S. carpocapsae ALL strain to date, comprising 84.5 Mb in 16 scaffolds, with an N50 of 7.36Mb. The largest scaffold, with 20.9Mb, was identified as chromosome X based on sex-specific genome sequencing. The high level of contiguity allowed us to characterize gene density, repeat content, and GC content. RNA-seq data from 17 developmental stages, spanning from embryo to adult, were used to predict 30,957 gene models. Using this new genome, we performed a macrosyntenic analysis to Caenorhabditis elegans and Pristionchus pacificus and found S. carpocapsae's chromosome X to be primarily orthologous to C. elegans' and P. pacificus' chromosome II and IV. We also investigated the expansion of protein families and gene expression differences between male and female stage nematodes. This new genome and more accurate set of annotations provide a foundation for new comparative genomic and gene expression studies within the Steinernema clade and across the Nematoda phylum.
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Sternberg, Paul, Mortazavi, Ali, Xian, Zhaoying, Mahanti, Parag, Zeng, Weihua, Hsueh, Yen-Ping, Gronquist, Matthew, Schwarz, Erich, Schroeder, Frank
[
International Worm Meeting,
2013]
Nematophagous fungi are natural predators of soil-dwelling nematodes, and this predator-prey relationship makes them an attractive model to study co-evolution. How do microorganisms detect their metazoan prey, and how do prey respond to predators? We set out to investigate the cues that nematophagous fungi use to trigger morphogenesis of their nematode-trapping devices and how C. elegans responds to nematophagous fungi. We found that nematophagous fungi can detect and respond to ascarosides, small molecules produced by many nematodes that regulate nematode development and behavior. In response to ascarosides, Arthrobotrys oligospora and closely related nematophagous fungi induce morphogenesis of their nematode traps. Ascarosides thus represent a conserved molecular pattern used by nematophagous fungi to detect prey. Through RNA-seq analysis, we identified A. oligospora genes regulated by ascarosides and nematode exposure. On the other hand, C. elegans are attracted to A. oligospora. This attraction is, at least in part, mediated by volatile compounds. Gas chromatography and mass spectrometry revealed that volatile organic compounds produced by A. oligospora could attract nematodes. Genetic analysis and cell-specific laser ablation showed that AWC neurons are required for this behavior. To find genes involved in the AWC-mediated A. oligospora attraction, we performed single-cell RNA-seq of the AWCon neuron. We detected expression of 6,608 genes in AWC neurons, with 1,278 being AWC-enriched. Preliminary mutant screening revealed genes that have a function in AWC-mediated chemosensation.