Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds

Comparative analysis of the transcriptome across distant species

The transcriptome is the readout of the genome. Identifying common features in it across distant species can reveal fundamental principles. To this end, the ENCODE and modENCODE consortia have generated large amounts of matched RNA-sequencing data for human, worm and fly. Uniform processing and comprehensive annotation of these data allow comparison across metazoan phyla, extending beyond earlier within-phylum transcriptome comparisons and revealing ancient, conserved features. Specifically, we discover co-expression modules shared across animals, many of which are enriched in developmental genes. Moreover, we use expression patterns to align the stages in worm and fly development and find a novel pairing between worm embryo and fly pupae, in addition to the embryo-to-embryo and larvae-to-larvae pairings. Furthermore, we find that the extent of non-canonical, non-coding transcription is similar in each organism, per base pair. Finally, we find in all three organisms that the gene-expression levels, both coding and non-coding, can be quantitatively predicted from chromatin features at the promoter using a 'universal model' based on a single set of organism-independent parameters

Mortality from gastrointestinal congenital anomalies at 264 hospitals in 74 low-income, middle-income, and high-income countries: a multicentre, international, prospective cohort study

Summary Background Congenital anomalies are the fifth leading cause of mortality in children younger than 5 years globally. Many gastrointestinal congenital anomalies are fatal without timely access to neonatal surgical care, but few studies have been done on these conditions in low-income and middle-income countries (LMICs). We compared outcomes of the seven most common gastrointestinal congenital anomalies in low-income, middle-income, and high-income countries globally, and identified factors associated with mortality. Methods We did a multicentre, international prospective cohort study of patients younger than 16 years, presenting to hospital for the first time with oesophageal atresia, congenital diaphragmatic hernia, intestinal atresia, gastroschisis, exomphalos, anorectal malformation, and Hirschsprung’s disease. Recruitment was of consecutive patients for a minimum of 1 month between October, 2018, and April, 2019. We collected data on patient demographics, clinical status, interventions, and outcomes using the REDCap platform. Patients were followed up for 30 days after primary intervention, or 30 days after admission if they did not receive an intervention. The primary outcome was all-cause, in-hospital mortality for all conditions combined and each condition individually, stratified by country income status. We did a complete case analysis. Findings We included 3849 patients with 3975 study conditions (560 with oesophageal atresia, 448 with congenital diaphragmatic hernia, 681 with intestinal atresia, 453 with gastroschisis, 325 with exomphalos, 991 with anorectal malformation, and 517 with Hirschsprung’s disease) from 264 hospitals (89 in high-income countries, 166 in middleincome countries, and nine in low-income countries) in 74 countries. Of the 3849 patients, 2231 (58·0%) were male. Median gestational age at birth was 38 weeks (IQR 36–39) and median bodyweight at presentation was 2·8 kg (2·3–3·3). Mortality among all patients was 37 (39·8%) of 93 in low-income countries, 583 (20·4%) of 2860 in middle-income countries, and 50 (5·6%) of 896 in high-income countries (p<0·0001 between all country income groups). Gastroschisis had the greatest difference in mortality between country income strata (nine [90·0%] of ten in lowincome countries, 97 [31·9%] of 304 in middle-income countries, and two [1·4%] of 139 in high-income countries; p≤0·0001 between all country income groups). Factors significantly associated with higher mortality for all patients combined included country income status (low-income vs high-income countries, risk ratio 2·78 [95% CI 1·88–4·11], p<0·0001; middle-income vs high-income countries, 2·11 [1·59–2·79], p<0·0001), sepsis at presentation (1·20 [1·04–1·40], p=0·016), higher American Society of Anesthesiologists (ASA) score at primary intervention (ASA 4–5 vs ASA 1–2, 1·82 [1·40–2·35], p<0·0001; ASA 3 vs ASA 1–2, 1·58, [1·30–1·92], p<0·0001]), surgical safety checklist not used (1·39 [1·02–1·90], p=0·035), and ventilation or parenteral nutrition unavailable when needed (ventilation 1·96, [1·41–2·71], p=0·0001; parenteral nutrition 1·35, [1·05–1·74], p=0·018). Administration of parenteral nutrition (0·61, [0·47–0·79], p=0·0002) and use of a peripherally inserted central catheter (0·65 [0·50–0·86], p=0·0024) or percutaneous central line (0·69 [0·48–1·00], p=0·049) were associated with lower mortality. Interpretation Unacceptable differences in mortality exist for gastrointestinal congenital anomalies between lowincome, middle-income, and high-income countries. Improving access to quality neonatal surgical care in LMICs will be vital to achieve Sustainable Development Goal 3.2 of ending preventable deaths in neonates and children younger than 5 years by 2030

Functional genomic analyses of development in mouse and regeneration in Hydra.

Gene expression at the transcriptional level is controlled by DNA sequences called cis-regulatory modules (CRM) and at the post-transcriptional level by microRNAs (miRNAs). CRMs have been studied almost exclusively in bilaterian organisms and little is known about them in non-bilaterian metazoans. Understanding the architecture of CRMs in cnidarians, a sister phylum to bilaterians, can potentially shed light on the evolution of gene regulation. Head regeneration is one of the most widely studied developmental processes in cnidarians. Using a comparison of the transcriptomes of regenerating heads and developing buds, I have determined sets of genes that are specific and common between head regeneration and budding. To understand the genomic sequences controlling these developmental programs, I have mapped the open-chromatin landscape of Hydra in different body parts and during head regeneration to identify candidate promoters and enhancers. My results are the first atlas of CRMs in Hydra, including a substantial fraction that is dynamic during head regeneration. Mammalian embryonic development has been used as a model system to study the role of miRNAs in previous studies, but a complete atlas of miRNA expression during development is missing. To understand the role of microRNAs during mouse development, I analyzed a time course of development representing multiple tissues and organs in mouse embryo. We find distinct tissue and developmental stage-specific miRNA expression profiles dominated by a small number of miRNAs. Analysis of conserved miRNAs reveals clustering of expression patterns by tissue types rather than species. We used matching RNA-seq and histone modification ChIP-seq datasets to improve the annotation of miRNA primary transcripts. We show that the expression levels of majority of primary miRNA transcripts predict the expression of their corresponding mature miRNAs. Our data provide the most comprehensive miRNA resource for mouse as well as a comprehensive list of mouse miRNAs that can be reliably measured by RNA-seq of their primary transcripts.Taken together, the elucidation of cis-regulatory landscape in the cnidarian Hydra and miRNA expression during mouse embryonic development will help the scientific community to understand better the role of enhancers in metazoan evolution and miRNA regulation in mammalian embryonic development

Functional genomic analyses of development in mouse and regeneration in Hydra.

Gene expression at the transcriptional level is controlled by DNA sequences called cis-regulatory modules (CRM) and at the post-transcriptional level by microRNAs (miRNAs). CRMs have been studied almost exclusively in bilaterian organisms and little is known about them in non-bilaterian metazoans. Understanding the architecture of CRMs in cnidarians, a sister phylum to bilaterians, can potentially shed light on the evolution of gene regulation. Head regeneration is one of the most widely studied developmental processes in cnidarians. Using a comparison of the transcriptomes of regenerating heads and developing buds, I have determined sets of genes that are specific and common between head regeneration and budding. To understand the genomic sequences controlling these developmental programs, I have mapped the open-chromatin landscape of Hydra in different body parts and during head regeneration to identify candidate promoters and enhancers. My results are the first atlas of CRMs in Hydra, including a substantial fraction that is dynamic during head regeneration. Mammalian embryonic development has been used as a model system to study the role of miRNAs in previous studies, but a complete atlas of miRNA expression during development is missing. To understand the role of microRNAs during mouse development, I analyzed a time course of development representing multiple tissues and organs in mouse embryo. We find distinct tissue and developmental stage-specific miRNA expression profiles dominated by a small number of miRNAs. Analysis of conserved miRNAs reveals clustering of expression patterns by tissue types rather than species. We used matching RNA-seq and histone modification ChIP-seq datasets to improve the annotation of miRNA primary transcripts. We show that the expression levels of majority of primary miRNA transcripts predict the expression of their corresponding mature miRNAs. Our data provide the most comprehensive miRNA resource for mouse as well as a comprehensive list of mouse miRNAs that can be reliably measured by RNA-seq of their primary transcripts.Taken together, the elucidation of cis-regulatory landscape in the cnidarian Hydra and miRNA expression during mouse embryonic development will help the scientific community to understand better the role of enhancers in metazoan evolution and miRNA regulation in mammalian embryonic development

Molecular evolution and expression of opsin genes in Hydra vulgaris.

BACKGROUND: The evolution of opsin genes is of great interest because it can provide insight into the evolution of light detection and vision. An interesting group in which to study opsins is Cnidaria because it is a basal phylum sister to Bilateria with much visual diversity within the phylum. Hydra vulgaris (H. vulgaris) is a cnidarian with a plethora of genomic resources to characterize the opsin gene family. This eyeless cnidarian has a behavioral reaction to light, but it remains unknown which of its many opsins functions in light detection. Here, we used phylogenetics and RNA-seq to investigate the molecular evolution of opsin genes and their expression in H. vulgaris. We explored where opsin genes are located relative to each other in an improved genome assembly and where they belong in a cnidarian opsin phylogenetic tree. In addition, we used RNA-seq data from different tissues of the H. vulgaris adult body and different time points during regeneration and budding stages to gain insight into their potential functions. RESULTS: We identified 45 opsin genes in H. vulgaris, many of which were located near each other suggesting evolution by tandem duplications. Our phylogenetic tree of cnidarian opsin genes supported previous claims that they are evolving by lineage-specific duplications. We identified two H. vulgaris genes (HvOpA1 and HvOpB1) that fall outside of the two commonly determined Hydra groups; these genes possibly have a function in nematocytes and mucous gland cells respectively. We also found opsin genes that have similar expression patterns to phototransduction genes in H. vulgaris. We propose a H. vulgaris phototransduction cascade that has components of both ciliary and rhabdomeric cascades. CONCLUSIONS: This extensive study provides an in-depth look at the molecular evolution and expression of H. vulgaris opsin genes. The expression data that we have quantified can be used as a springboard for additional studies looking into the specific function of opsin genes in this species. Our phylogeny and expression data are valuable to investigations of opsin gene evolution and cnidarian biology

Junonia coenia wing transcriptome

Assembled polyA mRNA-seq transcript fragments from Junonia coenia hindwings. Transcripts are from two polyphenic morphs (tan and red) across four developmental stages (late 5th instar, early pupa, ommochrome-stage pupa, early melanin-stage pupa)

Coordinated Gene Expression and Chromatin Regulation during Hydra Head Regeneration.

The cnidarian model organism Hydra has long been studied for its remarkable ability to regenerate its head, which is controlled by a head organizer located near the hypostome. The canonical Wnt pathway plays a central role in head organizer function during regeneration and during bud formation, which is the asexual mode of reproduction in Hydra. However, it is unclear how shared the developmental programs of head organizer genesis are in budding and regeneration. Time-series analysis of gene expression changes during head regeneration and budding revealed a set of 298 differentially expressed genes during the 48-h head regeneration and 72-h budding time courses. In order to understand the regulatory elements controlling Hydra head regeneration, we first identified 27,137 open-chromatin elements that are open in one or more sections of the organism body or regenerating tissue. We used histone modification ChIP-seq to identify 9,998 candidate proximal promoter and 3,018 candidate enhancer-like regions respectively. We show that a subset of these regulatory elements is dynamically remodeled during head regeneration and identify a set of transcription factor motifs that are enriched in the enhancer regions activated during head regeneration. Our results show that Hydra displays complex gene regulatory structures of developmentally dynamic enhancers, which suggests that the evolution of complex developmental enhancers predates the split of cnidarians and bilaterians

Hybrid Assembly of the Genome of the Entomopathogenic Nematode Steinernema carpocapsae Identifies the X-Chromosome.

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. carpocapsaes 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

Adapting the Smart-seq2 Protocol for Robust Single Worm RNA-seq.

Most nematodes are small worms that lack enough RNA for regular RNA-seq protocols without pooling hundred to thousand of individuals. We have adapted the Smart-seq2 protocol in order to sequence the transcriptome of an individual worm. While developed for individual Steinernema carpocapsae and Caenorhabditis elegans larvae as well as embryos, the protocol should be adaptable for other nematode species and small invertebrates. In addition, we describe how to analyze the RNA-seq results using the Galaxy online environment. We expect that this method will be useful for the studying gene expression variances of individual nematodes in wild type and mutant backgrounds