title: Efficient Enrichment of Bacterial mRNA from Host-Bacteria Total RNA Samples.
abstract: Despite numerous advances in genomics and bioinformatics, technological hurdles remain to examine host-microbe transcriptomics. Sometimes the transcriptome of either or both can be ascertained merely by generating more sequencing reads. However, many cases exist where bacterial mRNA needs to be enriched further to enable cost-effective sequencing of the pathogen or endosymbiont. While a suitable method is commercially available for mammalian samples of this type, development of such methods has languished for invertebrate samples. Furthermore, a common method across multiple taxa would facilitate comparisons between bacteria in invertebrate vectors and their vertebrate hosts. Here, a method is described to concurrently remove polyadenylated transcripts, prokaryotic rRNA, and eukaryotic rRNA, including those with low amounts of starting material (e.g. 100 ng). In a Wolbachia-Drosophila system, this bacterial mRNA enrichment yielded a 3-fold increase in Wolbachia mRNA abundance and a concomitant 3.3-fold increase in the percentage of transcripts detected. More specifically, 70% of the genome could be recovered by transcriptome sequencing compared to 21% in the total RNA. Sequencing of similar bacterial mRNA-enriched samples generated from Ehrlichia-infected canine cells covers 93% of the Ehrlichia genome, suggesting ubiquitous transcription across the entire Ehrlichia chaffeensis genome. This technique can potentially be used to enrich bacterial mRNA in many studies of host-microbe interactions.
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Nikhil
authors.initials: N
authors.lastname: Kumar
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, United States of America.
authors.forename: Mingqun
authors.initials: M
authors.lastname: Lin
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Xuechu
authors.initials: X
authors.lastname: Zhao
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Sandra
authors.initials: S
authors.lastname: Ott
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Ivette
authors.initials: I
authors.lastname: Santana-Cruz
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Sean
authors.initials: S
authors.lastname: Daugherty
authors.affiliation: Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210, United States of America.
authors.forename: Yasuko
authors.initials: Y
authors.lastname: Rikihisa
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Lisa
authors.initials: L
authors.lastname: Sadzewicz
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Luke J
authors.initials: LJ
authors.lastname: Tallon
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.|Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Claire M
authors.initials: CM
authors.lastname: Fraser
authors.affiliation: Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.|Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD 21201, United States of America.
authors.forename: Julie C
authors.initials: JC
authors.lastname: Dunning Hotopp
mesh_terms: D000818:Animals; D017178:Brugia malayi / Q000382:microbiology; D004330:Drosophila / Q000382:microbiology; D018469:Ehrlichia chaffeensis / Q000235:genetics; D005821:Genetic Techniques; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D054884:Host-Pathogen Interactions / Q000235:genetics*; D046210:Microfluidic Analytical Techniques; D011061:Poly A / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000737:chemistry / Q000302:isolation & purification*; D012335:RNA, Ribosomal / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013559:Symbiosis; D020577:Wolbachia / Q000235:genetics / Q000472:pathogenicity; D019596:Wolves / Q000382:microbiology
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D012335:RNA, Ribosomal; D011061:Poly A