The rise in throughput and quality of long-read sequencing should allow unambiguous identification of full-length transcript isoforms. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here we develop and characterize long-read Split-seq (LR-Split-seq), which uses combinatorial barcoding to sequence single cells with long reads. Applied to the C2C12 myogenic system, LR-split-seq associates isoforms to cell types with relative economy and design flexibility. We find widespread evidence of changing isoform expression during differentiation including alternative transcription start sites (TSS) and/or alternative internal exon usage. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells.
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 most complete genome of the S. carpocapsae ALL strain to date, comprising 84.5 Mb in 16 scaffolds, with an N50 of 7.36 Mb. The largest scaffold, with 20.9 Mb, 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 improved 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 adult male and female stage nematodes. This new genome and more accurate set of annotations provide a foundation for additional comparative genomic and gene expression studies within the Steinernema clade and across the Nematoda phylum.
Steinernema nematodes have been widely studied for insect infection and mutualism, but little is known about the patterns of gene expression along the body of these worms or how these compare to the model organism Caenorhabditis elegans. Here we perform the first comparative analysis between the heads and tail regions of Steinernema carpocapsae and C. elegans Infective Juveniles (IJs)/dauers and young adults using single-worm RNA-seq. While we find overall agreement in gene expression there were several sets of genes with substantial differences between the two species. Gene expression in the S. carpocapsae female compared to the C. elegans hermaphrodite heads and tails revealed differences in metabolism, aging, and determination of lifespan. Young adult male heads and tails showed major differences in developmental related processes such as morphogenesis as well as neuronal development and signaling. We also found head- and tail-specific gene expression differences between S. carpocapsae IJs and C. elegans dauers for genes related to growth and development as well as neuronal signaling and activity. This study is one of the first comparative transcriptomic analyses of body parts between distantly related species of nematodes and provides insight into both the highly conserved and genetically distinctive characteristics of both species.
Alternative RNA isoforms are defined by promoter choice, alternative splicing, and polyA site selection. Although differential isoform expression is known to play a large regulatory role in eukaryotes, it has proved challenging to study with standard short-read RNA-seq because of the uncertainties it leaves about the full-length structure and precise termini of transcripts. The rise in throughput and quality of long-read sequencing now makes it possible, in principle, to unambiguously identify most transcript isoforms from beginning to end. However, its application to single-cell RNA-seq has been limited by throughput and expense. Here, we develop and characterize long-read Split-seq (LR-Split-seq), which uses a combinatorial barcoding-based method for sequencing single cells and nuclei with long reads. We show that LR-Split-seq can associate isoforms with cell types with relative economy and design flexibility. We characterize LR-Split-seq for whole cells and nuclei by using the well-studied mouse C2C12 system in which mononucleated myoblast cells differentiate and fuse into multinucleated myotubes. We show that the overall results are reproducible when comparing long- and short-read data from the same cell or nucleus. We find substantial evidence of differential isoform expression during differentiation including alternative transcription start site (TSS) usage. We integrate the resulting isoform expression dynamics with snATAC-seq chromatin accessibility to validate TSS-driven isoform choices. LR-Split-seq provides an affordable method for identifying cluster-specific isoforms in single cells that can be further quantified with companion deep short-read scRNA-seq from the same cell populations.
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.Article SummaryThe insect killing worms Steinernema carpocapsae is a model organism for parasitism and symbiosis. The authors have used long reads and optical mapping to generate substantially contiguous assembly and a new set of gene annotations. They have identified the X chromosome as well as expansions in specific family proteases found in the venom of this worm. A macrosyntenic analysis with C. elegans shows a broad conservation of ancestral chromosomes with the exception of chromosome X. This new assembly will be useful to the Steinernema community and the broader nematode genomics community.
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