Preparation and Metatranscriptomic Analyses of Host–Microbe Systems

Hampton-Marcell, Jarrad; Moormann, Stephanie M.; Owens, Sarah M.; Gilbert, Jack A.

doi:10.1016/b978-0-12-407863-5.00009-5

Cited by 17 publications

(14 citation statements)

References 9 publications

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“…Dual sequencing of the host and pathogen transcriptomes offers an extraordinary opportunity to study molecular interactions occurring during a productive infection, with the advantage of avoiding the typical biases of other experimental approaches (Westermann et al ., ; Hampton‐Marcell et al ., ).…”

Section: Discussionmentioning

confidence: 97%

Dual analysis of host and pathogen transcriptomes in ostreid herpesvirus 1‐positive Crassostrea gigas

Rosani

Varotto

Domeneghetti

et al. 2014

Environmental Microbiology

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Ostreid herpesvirus type 1 (OsHV-1) has become a problematic infective agent for the Pacific oyster Crassostrea gigas. In particular, the OsHV-1 μVar subtype has been associated with severe mortality episodes in oyster spat and juvenile oysters in France and other regions of the world. Factors enhancing the infectivity of the virus and its interactions with susceptible and resistant bivalve hosts are still to be understood, and only few studies have explored the expression of oyster or viral genes during productive infections. In this work, we have performed a dual RNA sequencing analysis on an oyster sample with a high viral load. High sequence coverage allowed us to thoroughly explore the OsHV-1 transcriptome and identify the activated molecular pathways in C. gigas. The identification of several highly induced and defence-related oyster transcripts supports the crucial role played by the innate immune system against the virus and opportunistic microbes possibly contributing to subsequent spat mortality.

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Section: Discussionmentioning

confidence: 97%

Dual analysis of host and pathogen transcriptomes in ostreid herpesvirus 1‐positive Crassostrea gigas

Rosani

Varotto

Domeneghetti

et al. 2014

Environmental Microbiology

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“…Our strategy for enrichment of prokaryotic mRNA consisted of subtracting eukaryotic mRNA and rRNA from total host‐symbiont RNA, which allowed us to produce metatranscriptomes that were enriched in prokaryotic functional gene transcripts. This strategy is similar to the protocol from Hampton‐Marcell and colleagues (), and it should be applicable for the isolation of any host‐associated microbial metatranscriptomes. Furthermore, the isolated eukaryotic mRNA would be suitable for sequencing, making it possible to obtain complementary host and symbiont transcriptome pairs.…”

Section: Discussionmentioning

confidence: 99%

Revealing microbial functional activities in the Red Sea sponge Stylissa carteri by metatranscriptomics

Moitinho‐Silva¹,

Seridi

Ryu

et al. 2014

Environmental Microbiology

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Sponges are important components of marine benthic environments and are associated with microbial symbionts that carry out ecologically relevant functions. Stylissa carteri is an abundant, low-microbial abundance species in the Red Sea. We aimed to achieve the functional and taxonomic characterization of the most actively expressed prokaryotic genes in S. carteri. Prokaryotic mRNA was enriched from sponge total RNA, sequenced using Illumina HiSeq technology and annotated using the metagenomics Rapid Annotation using Subsystem Technology (MG-RAST) pipeline. We detected high expression of archaeal ammonia oxidation and photosynthetic carbon fixation by members of the genus Synechococcus. Functions related to stress response and membrane transporters were among the most highly expressed by S. carteri symbionts. Unexpectedly, gene functions related to methylotrophy were highly expressed by gammaproteobacterial symbionts. The presence of seawater-derived microbes is indicated by the phylogenetic proximity of organic carbon transporters to orthologues of members from the SAR11 clade. In summary, we revealed the most expressed functions of the S. carteri-associated microbial community and linked them to the dominant taxonomic members of the microbiome. This work demonstrates the applicability of metatranscriptomics to explore poorly characterized symbiotic consortia and expands our knowledge of the ecologically relevant functions carried out by coral reef sponge symbionts.

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“…RNA quality was assessed via RNA integrity number (RIN) values obtained using Agilent Bioanalyzer software (average RIN = 6.4; n =6). Metatranscriptome libraries were generated following standard protocols in the Argonne NGS core laboratory at the University of Chicago 37 . Completed metatranscriptome libraries were quality checked on the Agilent Bioanalyzer, quantified using Qubit (Invitrogen, Carlsbad, CA), diluted to 2 nM and denatured for sequencing on the Illumina MiSeq.…”

Section: Methodsmentioning

confidence: 99%

Genetic deletion of the bacterial sensor NOD2 improves murine Crohn's disease-like ileitis independent of functional dysbiosis

et al. 2017

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Although genetic polymorphisms in NOD2 (nucleotide-binding oligomerization domain-containing 2) have been associated with the pathogenesis of Crohn’s disease (CD), little is known regarding the role of wild-type (WT) NOD2 in the gut. To date, most murine studies addressing the role of WT Nod2 have been conducted using healthy (ileitis/colitis-free) mouse strains. Here, we evaluated the effects of Nod2 deletion in a murine model of spontaneous ileitis, i.e., the SAMP1Yit/Fc (SAMP) strain, which closely resembles CD. Remarkably, Nod2 deletion improved both chronic cobblestone ileitis (by 50% assessed, as the % of abnormal mucosa at 24 wks of age), as well as acute dextran sodium sulfate (DSS) colitis. Mechanistically, Th2 cytokine production and Th2-transcription factor activation (i.e., STAT6 phosphorylation) were reduced. Microbiologically, the effects of Nod2 deletion appeared independent of fecal microbiota composition and function, assessed by 16S rRNA and metatranscriptomics. Our findings indicate that pharmacological blockade of NOD2 signaling in humans could improve health in Th2-driven chronic intestinal inflammation.

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Preparation and Metatranscriptomic Analyses of Host–Microbe Systems

Cited by 17 publications

References 9 publications

Dual analysis of host and pathogen transcriptomes in ostreid herpesvirus 1‐positive Crassostrea gigas

Dual analysis of host and pathogen transcriptomes in ostreid herpesvirus 1‐positive Crassostrea gigas

Revealing microbial functional activities in the Red Sea sponge Stylissa carteri by metatranscriptomics

Genetic deletion of the bacterial sensor NOD2 improves murine Crohn's disease-like ileitis independent of functional dysbiosis

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