Mortazavi A et al. (2010) Genome Res "Scaffolding a Caenorhabditis nematode genome with RNA-seq."

Wold BJ, Schwarz EM, Williams B, Schaeffer L, Sternberg PW, Mortazavi A, Antoshechkin I

[Genome Res, 2010]

Efficient sequencing of animal and plant genomes by next-generation technology should allow many neglected organisms of biological and medical importance to be better understood. As a test case, we have assembled a draft genome of Caenorhabditis sp. 3 PS1010 through a combination of direct sequencing and scaffolding with RNA-seq. We first sequenced genomic DNA and mixed-stage cDNA using paired 75-nt reads from an Illumina GAII. A set of 230 million genomic reads yielded an 80-Mb assembly, with a supercontig N50 of 5.0 kb, covering 90% of 429 kb from previously published genomic contigs. Mixed-stage poly(A)(+) cDNA gave 47.3 million mappable 75-mers (including 5.1 million spliced reads), which separately assembled into 17.8 Mb of cDNA, with an N50 of 1.06 kb. By further scaffolding our genomic supercontigs with cDNA, we increased their N50 to 9.4 kb, nearly double the average gene size in C. elegans. We predicted 22,851 protein-coding genes, and detected expression in 78% of them. Multigenome alignment and data filtering identified 2672 DNA elements conserved between PS1010 and C. elegans that are likely to encode regulatory sequences or previously unknown ncRNAs. Genomic and cDNA sequencing followed by joint assembly is a rapid and useful strategy for biological analysis.

Mortazavi, Ali et al. (2013) International Worm Meeting "Genomic analysis of Steinernema: Insights into insect parasitism, intragenus and intergenus evolution."

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.

Mortazavi, Ali et al. (2009) International Worm Meeting "Ultra-high throughput sequencing of the genome and transcriptome of Caenorhabditis sp. 3 PS1010."

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.

Liang, H. et al. (2019) International Worm Meeting "Chromatin Accessibility Changes Discovery on S. Carpocapsae using ATAC-seq and Method Replication on C. elegans."

Mortazavi, A., Liang, H.

[International Worm Meeting, 2019]

Steinernema Carpocapsae (S. carpocapsae) is a entomopathogenic nematode (EPN) that is widely known to be a "natural insecticide". The infective juvenile (IJ) infect and reproduce in their insect hosts and kill them at their larvae stage together with specific symbiotic bacteria. We are interested in understanding the changes in chromatin openness of S. carpocapsae during development using ATAC-seq and comparing the results to matching stages in C. elegans. We are currently optimizing our protocol in L1 worms and hope to apply to other stages. After successfully replicating ATAC-seq on L1 at arrest of C. elegans and the matching stage of S. Carpocapsae, further comparative genomic in all stages can be studied to identify the conserved regulatory elements between the two species.

Duan, Ye et al. (2020) MicroPubl Biol

Ambros, Victor, Sun, Yongming, Duan, Ye

[MicroPubl Biol, 2020]

RNA-seq is widely used for the quantitative analysis of transcriptomes in the context of studies of gene expression and regulation (Mortazavi et al., 2008; Ozsolak and Milos, 2011; Wang et al., 2009). Generally, RNA-seq protocols employ poly(A) selection for mRNA enrichment. However, poly(A) based enrichment is subject to potential bias depending on the poly(A) status of various mRNAs, which could be particularly undesirable in the context of studying post-transcriptional gene regulatory mechanisms, such miRNA repression (Wu et al., 2006). Therefore, ribosomal RNA (rRNA) depletion is a desirable alternative strategy to enrich for mRNA sequences in RNA-seq sample preparation (Zhao et al., 2014). However, currently available rRNA depletion toolkits were designed for either mammals or bacteria, and hence do not offer an efficient option for rRNA depletion of RNA samples from certain experimental organisms, such as C. elegans.

Dillman AR et al. (2012) J Nematol "Incorporating genomics into the toolkit of nematology."

Mortazavi A, Sternberg PW, Dillman AR

[J Nematol, 2012]

The study of nematode genomes over the last three decades has relied heavily on the model organism Caenorhabditis elegans, which remains the best-assembled and annotated metazoan genome. This is now changing as a rapidly expanding number of nematodes of medical and economic importance have been sequenced in recent years. The advent of sequencing technologies to achieve the equivalent of the $1000 human genome promises that every nematode genome of interest will eventually be sequenced at a reasonable cost. As the sequencing of species spanning the nematode phylum becomes a routine part of characterizing nematodes, the comparative approach and the increasing use of ecological context will help us to further understand the evolution and functional specializations of any given species by comparing its genome to that of other closely and more distantly related nematodes. We review the current state of nematode genomics and discuss some of the highlights that these genomes have revealed and the trend and benefits of ecological genomics, emphasizing the potential for new genomes and the exciting opportunities this provides for nematological studies.

McGill, C.J. et al. (2019) International Worm Meeting "Variations in microRNA expression during development of divergent Caenorhabditis elegans strains."

Mortazavi, A., Serra, L., Rodriguez, I.M, McGill, C.J.

[International Worm Meeting, 2019]

MicroRNAs(miRNAs) are small, 17-22nt, non-coding RNAs that play a critical role in post-transcriptional regulation including during development. We are interested in studying the impact of microRNA expression variation in Caenorhabditis elegans strains by characterizing the expression of miRNAs and mRNA at the L1 stage using twelve Caenorhabditis elegans Natural Diversity Resource (CeNDR) strains that constitute the "divergent set". Our goal is to investigate variations in miRNA and mRNA profiles at the L1 stage in response to developmental stress. Preliminary results indicate that each strain has a distinct gene expression profile. We also found that N2 expresses more dauer related genes compared to CB4856. We will compare eleven CeNDR strains to N2 and identify key differences in miRNAs and mRNA expression.

Rodriguez, B.C. et al. (2017) International Worm Meeting "Transcriptomic analysis of post-embryonic development across two distantly related nematode genera."

Mortazavi, A., Serra, L., Macchietto, M., Rodriguez, B.C.

[International Worm Meeting, 2017]

Entomopathogenic nematodes (EPNs) are parasitic nematodes that can efficiently kill insects and are primarily composed of two intensively studied genera: Steinernema and Heterohabditis. EPNs and Caenorhabditis elegans last common ancestor was approximately 200 MYA. Observations comparing both nematodes affirm their morphological similarities. However, when examined closely, their reproduction, cellular structures, and living preferences are distinguished. Steinernema carpocapsae are gonochoristic; they have a female and male. On the other hand, Caenorhabditis elegans is hermaphroditic and male. We are interested in characterizing sex determination in Steinernema carpocapsae and comparing the conservation of sex determining genes to C. elegans. We are comparing the post-embryonic developmental stages larvae 1, larvae 2, larvae 3, larvae 4, infective juvenile, young adult male and female gene expression at the single-worm level to Caenorhabditis elegans. to assay gene expression at each stage by using the Smart-Seq2 protocol, which amplifies small quantities of mRNA from the nematodes into cDNA for sequencing. The S. carpocapsae samples were analyzed to examine correlation and quality of samples. C. elegans samples will be collected to give a complete picture of the gene expression profiles and how each stage compares to the other organism. We are particularly interested in the sexes of both nematode species since they have similarities (both have males) and differences (C. elegans has hermaphrodites and S. carpocapsae has females).We have sequenced single worms for L1, infective juvenile, adult male and female for S. carpocapsae. We are isolating miRNA for each of the listed stages to enrich the analysis and have a complete assessment of the pathways involved in determining male and females in S. carpocapsae. We will then compare at single-worm level the conservation of sex determining genes to C. elegans.

McGill, C.J. et al. (2017) International Worm Meeting "Comparing miRNAs across developmental stages of C. elegans and other nematodes."

Macchietto, M., Phan, J.L., McGill, C.J., Serra, L., Mortazavi, A., Rodriguez, B.

[International Worm Meeting, 2017]

MicroRNAs(miRNAs) are small,17-22bp, non-coding RNAs that have been shown to have a significant regulatory effect on gene expression. Measuring this regulation is vital to understanding how gene expression progresses through developmental programs. We are interested in studying this through the Caenorhabditis elegans developmental model system. Due to older technologies, previous studies had to be done on thousands of worms at a time, and thus, the resolution was poor. New technology has enabled us to cut that number to few worms, which has drastically improved the resolution. We, also, will be profiling miRNAs at every stage of development to study the progression of miRNAs. We will compare our results to other nematodes such as Steinernema carpocapsae and Caenorhabditi angaria. This will be the first comparison of miRNAs expression at the single-worm level among distant nematode species with a focus on miRNAs involved in gene regulation during development.

Rodriguez, I. et al. (2019) International Worm Meeting "Comparative Transcriptomics of heads and tails between Steinernema carpocapsae and Caenorhabditis elegans."

Rodriguez, I., McGill, C., Rodriguez, B., Mortazavi, A., Serra, L.

[International Worm Meeting, 2019]

Entomopathogenic nematodes (EPNs) are parasitic nematodes that can efficiently kill insects, with Steinernema carpocapsae being the most studied EPN. Although Steinernematids have been widely studied for insect infection and mutualism and their commercial applications are already established, little is known about the patterns of gene expression in heads and tails of infective juveniles (IJs) and adults or how these compare to expression in the model organism C. elegans. Comparisons between Steinernema and Caenorhabditis demonstrate morphological similarities such as shape and morphology of the body, mouth cavity, pharynx and digestive system, although C. elegans is hermaphroditic while S. carpocapsae is gonochoristic, i.e. has males and females. While males of both S. carpocapsae and C. elegans have an additional set of neurons in the lower tail region that control mating behavior, the tail morphology is also very distinct. We hypothesize that there is a core set of sex-specific genes that are co-expressed in both S. carpocapsae and C. elegans adult sexes in a region specific manner. We performed a comparative analysis between heads and tails of S. carpocapsae and C. elegans IJs/dauers as well as male, female/hermaphrodite adults using single-worm RNA-seq. We have found that male tails of S. carpocapsae have similar gene expression to C. elegans. This is one of the first comparative transcriptomic analyses of body parts between distantly related species of nematodes and provides insights into both the highly conserved and genetically distinctive characteristics of both species.