title: Variation in RNA-Seq transcriptome profiles of peripheral whole blood from healthy individuals with and without globin depletion.
abstract: BACKGROUND
  The molecular profile of circulating blood can reflect physiological and pathological events occurring in other tissues and organs of the body and delivers a comprehensive view of the status of the immune system. Blood has been useful in studying the pathobiology of many diseases. It is accessible and easily collected making it ideally suited to the development of diagnostic biomarker tests. The blood transcriptome has a high complement of globin RNA that could potentially saturate next-generation sequencing platforms, masking lower abundance transcripts. Methods to deplete globin mRNA are available, but their effect has not been comprehensively studied in peripheral whole blood RNA-Seq data. In this study we aimed to assess technical variability associated with globin depletion in addition to assessing general technical variability in RNA-Seq from whole blood derived samples.
  
  
  RESULTS
  We compared technical and biological replicates having undergone globin depletion or not and found that the experimental globin depletion protocol employed removed approximately 80% of globin transcripts, improved the correlation of technical replicates, allowed for reliable detection of thousands of additional transcripts and generally increased transcript abundance measures. Differential expression analysis revealed thousands of genes significantly up-regulated as a result of globin depletion. In addition, globin depletion resulted in the down-regulation of genes involved in both iron and zinc metal ion bonding.
  
  
  CONCLUSIONS
  Globin depletion appears to meaningfully improve the quality of peripheral whole blood RNA-Seq data, and may improve our ability to detect true biological variation. Some concerns remain, however. Key amongst them the significant reduction in RNA yields following globin depletion. More generally, our investigation of technical and biological variation with and without globin depletion finds that high-throughput sequencing by RNA-Seq is highly reproducible within a large dynamic range of detection and provides an accurate estimation of RNA concentration in peripheral whole blood. High-throughput sequencing is thus a promising technology for whole blood transcriptomics and biomarker discovery.
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Medicine (Division of Respiratory Medicine), University of British Columbia, Vancouver, British Columbia, Canada; UBC James Hogg Research Centre & Institute for HEART + LUNG Health, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Heesun
authors.initials: H
authors.lastname: Shin
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada.
authors.forename: Casey P
authors.initials: CP
authors.lastname: Shannon
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Statistics, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Nick
authors.initials: N
authors.lastname: Fishbane
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC James Hogg Research Centre & Institute for HEART + LUNG Health, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Jian
authors.initials: J
authors.lastname: Ruan
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada.
authors.forename: Mi
authors.initials: M
authors.lastname: Zhou
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Statistics, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Robert
authors.initials: R
authors.lastname: Balshaw
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada.
authors.forename: Janet E
authors.initials: JE
authors.lastname: Wilson-McManus
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Computer Science, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Raymond T
authors.initials: RT
authors.lastname: Ng
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada; UBC James Hogg Research Centre & Institute for HEART + LUNG Health, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Bruce M
authors.initials: BM
authors.lastname: McManus
authors.affiliation: NCE CECR PROOF Centre of Excellence, Vancouver, British Columbia, Canada; UBC Department of Medicine (Division of Respiratory Medicine), University of British Columbia, Vancouver, British Columbia, Canada; UBC James Hogg Research Centre & Institute for HEART + LUNG Health, University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Scott J
authors.initials: SJ
authors.lastname: Tebbutt
mesh_terms: D001798:Blood Proteins / Q000235:genetics*; D005260:Female; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D005914:Globins / Q000737:chemistry* / Q000172:deficiency; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D058977:Molecular Sequence Annotation; D012333:RNA, Messenger / Q000097:blood*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D059467:Transcriptome
chemical_list: D001798:Blood Proteins; D012333:RNA, Messenger; D005914:Globins