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Glue Grant Human Transcriptome Array

Glue Grant Human Transcriptome Array. PNAS 2011 108 (9) 3707-3712; published ahead of print February 11, 2011, doi:10.1073/pnas.1019753108. Goals of the study.

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Glue Grant Human Transcriptome Array

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  1. Glue Grant Human Transcriptome Array

  2. PNAS 2011 108 (9) 3707-3712; published ahead of print February 11, 2011, doi:10.1073/pnas.1019753108

  3. Goals of the study • Evaluate the performance of Human Transcriptome array in terms of coverage, reproducibility, detection power, throughput, and cost effectiveness • Compare RNA-seq vs microarrays I

  4. Human Transcriptome Array design

  5. 10 probes/PSR • 119 probes/gene • 4 probes/exon-exon junction

  6. Array Control Probes

  7. Comparative analysis of RNA-seq and arrays

  8. Reproducibility:Microarray vs ~46 million mapped reads “The observed lower reproducibility is most obvious with low-abundance exons in which a difference of one or two reads between repeats can induce a large variance in the estimation.” Pearson correlation Microarray yields high quality data cost effectively

  9. Microarrays have lower variance over a larger range • “Both sample preparation and sequencing step contribute to the observed variance”

  10. Lower abundant transcripts are better detected by arrays • Low abundant transcripts have important clinical implication • Immune mediators • IFNs • IL • Chemokines Low Transcript abundance High Xu W. PNAS 2011 108 (9) 3707-3712;

  11. Required reads to cover detected probesets on the HTA Covered by less than 20 reads.

  12. Microarray covers the majority of junctions ID by RNA-seq with high confidence

  13. Array Design and Analysis

  14. Defining the Transcriptome • Designing an array requires a deconstruction of vast amounts of bioinformatic information followed by a reassembly into canonical gene representations • Sources of input • Ensembl (38) • Refseq (hNCBI36) • ExonWalk (hNCBI36_walk) • UCSC Known gene (hg18)

  15. Example: Leptin Receptor Well Annotated Transcript Variation and Transcript Redundancy

  16. Alternative Splicing and Transcript Diversity Example of a gene with 4 known transcript isoforms.

  17. Evidence for Alternative Splicing Event • 4 pieces of evidence for alternative splicing event • Different level of expression for the exon • Different level of expression for 2 adjacent junction probe sets • Different level of expression for 1 “exon skipping” probe set A C A B C

  18. Assay and Array Requirements • Modified WT 2.0 Assay • Input 50ng Total RNA • Assay produces double stranded labeled product • Standard cartridge • Hyb Oven • Fluidics • GCS3000 Scanner

  19. Modified Whole Transcript Assay(Double Stranded) cRNA from Ambion WT Expression Kit 2nd Cycle, 1st strand synthesis using SSII 2nd Cycle, 2nd strand synthesis using RNaseH and Klenow 3’→5’ exonuclease Divide into 3 RXNs Frag and Label using Affymetrix Terminal Labeling kit Recombine into 1 and concentrate HWS

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