glue grant human transcriptome array. 2 affymetrix confidential pnas 2011 108 (9) 3707-3712;...
TRANSCRIPT
Glue Grant Human Transcriptome Array
2Affymetrix Confidential
PNAS 2011 108 (9) 3707-3712; published ahead of printFebruary 11, 2011, doi:10.1073/pnas.1019753108
3Affymetrix 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
4Affymetrix Confidential
Human Transcriptome Array design
5Affymetrix Confidential
• 10 probes/PSR• 119 probes/gene• 4 probes/exon-exon junction
6Affymetrix Confidential
Array Control Probes
7Affymetrix Confidential
COMPARATIVE ANALYSIS OF RNA-SEQ AND ARRAYS
8Affymetrix 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
9Affymetrix Confidential
Microarrays have lower variance over a larger range
Exon Gene
Microarray 0.06 0.11
RNA-seq 0.12 0.31
Percent with lower CVs on array 85% 80%
“Both sample preparation and sequencing step contribute to the observed variance”
10Affymetrix 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;
11Affymetrix Confidential
Required reads to cover detected probesets on the HTA
Covered by less than 20 reads.
Reads Exon Gene
4M 90% 56%
11M 80% 46%
39M 60% 35%
12Affymetrix Confidential
Microarray covers the majority of junctions ID by RNA-seq with high confidence
13Affymetrix Confidential
ARRAY DESIGN AND ANALYSIS
14Affymetrix 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 inputEnsembl (38)Refseq (hNCBI36)ExonWalk (hNCBI36_walk)UCSC Known gene (hg18)
15Affymetrix Confidential
Example: Leptin Receptor
Well Annotated Transcript Variation and Transcript Redundancy
16Affymetrix Confidential
Alternative Splicing and Transcript Diversity
1 2 3 4 5 6 7 8 9
Example of a gene with 4 known transcript isoforms.
17Affymetrix 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
A CCA B
19Affymetrix 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
20Affymetrix Confidential
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