glue grant human transcriptome array. 2 affymetrix confidential pnas 2011 108 (9) 3707-3712;...

Glue Grant Human Transcriptome Array

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

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• 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

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Human Transcriptome Array design

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

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Array Control Probes

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COMPARATIVE ANALYSIS OF RNA-SEQ AND ARRAYS

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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

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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”

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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;

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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%

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

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ARRAY DESIGN AND ANALYSIS

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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)

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Example: Leptin Receptor

Well Annotated Transcript Variation and Transcript Redundancy

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Alternative Splicing and Transcript Diversity

1 2 3 4 5 6 7 8 9

Example of a gene with 4 known transcript isoforms.

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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

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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

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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