title: 3' tag digital gene expression profiling of human brain and universal reference RNA using Illumina Genome Analyzer.
abstract: BACKGROUND
  Massive parallel sequencing has the potential to replace microarrays as the method for transcriptome profiling. Currently there are two protocols: full-length RNA sequencing (RNA-SEQ) and 3'-tag digital gene expression (DGE). In this preliminary effort, we evaluated the 3' DGE approach using two reference RNA samples from the MicroArray Quality Control Consortium (MAQC).
  
  
  RESULTS
  Using Brain RNA sample from multiple runs, we demonstrated that the transcript profiles from 3' DGE were highly reproducible between technical and biological replicates from libraries constructed by the same lab and even by different labs, and between two generations of Illumina's Genome Analyzers. Approximately 65% of all sequence reads mapped to mitochondrial genes, ribosomal RNAs, and canonical transcripts. The expression profiles of brain RNA and universal human reference RNA were compared which demonstrated that DGE was also highly quantitative with excellent correlation of differential expression with quantitative real-time PCR. Furthermore, one lane of 3' DGE sequencing, using the current sequencing chemistry and image processing software, had wider dynamic range for transcriptome profiling and was able to detect lower expressed genes which are normally below the detection threshold of microarrays.
  
  
  CONCLUSIONS
  3' tag DGE profiling with massive parallel sequencing achieved high sensitivity and reproducibility for transcriptome profiling. Although it lacks the ability of detecting alternative splicing events compared to RNA-SEQ, it is much more affordable and clearly out-performed microarrays (Affymetrix) in detecting lower abundant transcripts.
authors.affiliation: Division of Biomedical Statistics and Informatics, Department of Health Sciences Research, Mayo Clinic, Rochester, MN 55905, USA.
authors.forename: Yan W
authors.initials: YW
authors.lastname: Asmann
authors.forename: Eric W
authors.initials: EW
authors.lastname: Klee
authors.forename: E Aubrey
authors.initials: EA
authors.lastname: Thompson
authors.forename: Edith A
authors.initials: EA
authors.lastname: Perez
authors.forename: Sumit
authors.initials: S
authors.lastname: Middha
authors.forename: Ann L
authors.initials: AL
authors.lastname: Oberg
authors.forename: Terry M
authors.initials: TM
authors.lastname: Therneau
authors.forename: David I
authors.initials: DI
authors.lastname: Smith
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Poland
authors.forename: Eric D
authors.initials: ED
authors.lastname: Wieben
authors.forename: Jean-Pierre A
authors.initials: JP
authors.lastname: Kocher
mesh_terms: D001921:Brain / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods* / Q000592:standards*; D015894:Genome, Human / Q000235:genetics*; D023281:Genomics / Q000379:methods* / Q000592:standards*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis; D016133:Polymerase Chain Reaction; D012313:RNA / Q000235:genetics*; D012015:Reference Standards; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D012984:Software; D013997:Time Factors
chemical_list: D012313:RNA