1. Glue Grant Human Transcriptome Array

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

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

4. 4 Affymetrix Confidential Human Transcriptome Array design

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

6. 6 Affymetrix Confidential Array Control Probes

7. 7 Affymetrix Confidential COMPARATIVE ANALYSIS OF RNA-SEQ AND ARRAYS

8. 8 Affymetrix Confidential 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. 9 Affymetrix Confidential Microarrays have lower variance over a larger range ExonGene Microarray0.060.11 RNA-seq0.120.31 Percent with lower CVs on array 85%80% “Both sample preparation and sequencing step contribute to the observed variance”

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

11. 11 Affymetrix Confidential Required reads to cover detected probesets on the HTA Covered by less than 20 reads. ReadsExonGene 4M90%56% 11M80%46% 39M60%35%

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

13. 13 Affymetrix Confidential ARRAY DESIGN AND ANALYSIS

14. 14 Affymetrix Confidential 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. 15 Affymetrix Confidential Example: Leptin Receptor Well Annotated Transcript Variation and Transcript Redundancy

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

17. 17 Affymetrix Confidential 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 AC CAB

18. 18 Affymetrix Confidential Detection of alternative splicing  Detection of alternative splicing events using exon and junction probes on the array  Two isoforms of SLK are alternatively spliced between liver and muscle; the green lines represent an isoform-skipping exon 15 and the blue lines represent another isoform including exon 15  (A) Changes of the signal of junction and exon probes  (B) Changes of the calculated expression of exons and junctions  Exon expression is shown at the diagonal, and junction expression is shown off the diagonal of two connecting exons

19. 19 Affymetrix Confidential 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

20. 20 Affymetrix Confidential Modified Whole Transcript Assay (Double Stranded) cRNA from Ambion WT Expression Kit 2 nd Cycle, 1 st strand synthesis using SSII 2 nd Cycle, 2 nd 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