title: Enriching pathogen transcripts from infected samples: a capture-based approach to enhanced host-pathogen RNA sequencing.
abstract: To fully understand the interactions of a pathogen with its host, it is necessary to analyze the RNA transcripts of both the host and pathogen throughout the course of an infection. Although this can be accomplished relatively easily on the host side, the analysis of pathogen transcripts is complicated by the overwhelming amount of host RNA isolated from an infected sample. Even with the read depth provided by second-generation sequencing, it is extremely difficult to get enough pathogen reads for an effective gene-level analysis. In this study, we describe a novel capture-based technique and device that considerably enriches for pathogen transcripts from infected samples. This versatile method can, in principle, enrich for any pathogen in any infected sample. To test the technique's efficacy, we performed time course tissue culture infections using Rift Valley fever virus and Francisella tularensis. At each time point, RNA sequencing (RNA-Seq) was performed and the results of the treated samples were compared with untreated controls. The capture of pathogen transcripts, in all cases, led to more than an order of magnitude enrichment of pathogen reads, greatly increasing the number of genes hit, the coverage of those genes, and the depth at which each transcript was sequenced.
authors.affiliation: Sandia National Laboratories, Livermore, CA 94551, USA.
authors.forename: Zachary W
authors.initials: ZW
authors.lastname: Bent
authors.forename: Mary B
authors.initials: MB
authors.lastname: Tran-Gyamfi
authors.forename: Stanley A
authors.initials: SA
authors.lastname: Langevin
authors.forename: David M
authors.initials: DM
authors.lastname: Brazel
authors.forename: Rachelle Y
authors.initials: RY
authors.lastname: Hamblin
authors.forename: Steven S
authors.initials: SS
authors.lastname: Branda
authors.forename: Kamlesh D
authors.initials: KD
authors.lastname: Patel
authors.forename: Todd W
authors.initials: TW
authors.lastname: Lane
authors.forename: Victoria A
authors.initials: VA
authors.lastname: VanderNoot
mesh_terms: D002460:Cell Line; D005604:Francisella tularensis / Q000235:genetics* / Q000502:physiology*; D020869:Gene Expression Profiling; D054884:Host-Pathogen Interactions; D006801:Humans; D008264:Macrophages / Q000382:microbiology / Q000821:virology; D009693:Nucleic Acid Hybridization; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D012296:Rift Valley fever virus / Q000235:genetics* / Q000502:physiology*; D017423:Sequence Analysis, RNA / Q000379:methods*
chemical_list: D012329:RNA, Bacterial; D012333:RNA, Messenger; D012367:RNA, Viral