This document summarizes the results of five phases of testing ERCC control transcripts on Agilent microarrays to evaluate their performance. In Phase III, ERCC transcripts were spiked into background RNA at various dilutions. Analysis of probe performance at different distances from the 3' end showed good signals for probes >1000nt from the polyA tail. Phase IV constructed pools with ERCCs at different concentrations to test a wide dynamic range. Phase V experiments showed Agilent microarrays can detect small differences in ERCC levels used as barcodes to track samples. The ERCC controls helped evaluate the microarray process and different platforms.

1. ERCC Transcripts Provide
Confidence to the
Performance of Agilent
One-Color and Two-Color
Microarray Experiments
Anne Bergstrom Lucas
Senior Research Scientist
July 10th 2014

2. Overview
- Quick review of ERCC Phases
- Agilent microarray probe generation and assay overview
- Questions regarding the dilution of the ERCC transcript pools
- Probe/ERCC performance in Phase III (latin-square proof-of-concept)
- Probe/ERCC performance in Phase IV (functional testing)
- Probe/ERCC performance in Phase V (external validation)
- Conclusions
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3. Number of ERCC clones
177
144
106
97
96
The Five Phases of ERCC Control Testing
BMC Genomics 2005, 6:150
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4. ERCC Control Transcripts Test Entire Assay
Spike-in controls for Validation
External, Exogenous
Sequences that do not
naturally appear in sample
Synthetic
Known [RNA]
traceable to SI
Known Sequence
traceable to reference
library RM
Track “whole-system” technical
performance
RNA
Isolation
Target
Preparation
optional mRNA
Amplification
Hybridization
Array
Content
Hybridized
Target
Detection
Expression
Measures
Biostatistical
Analysis
Spike-ins
to track
whole-
process
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5. ERCC transcripts were uploaded into the Agilent eArray program:
• Ran probe selection program using two different modes:
- “Best Probe”
- “Best Distribution”
Combined the lists from each mode to give ten probes that span each control sequence up to
>1000 bases from the 3’ end
- Two copies of each of the ten probes were ported into the 8x15K microarray format
- The rest of the space was filled with human biological genes from the catalog array
Used eArray Program for ERCC Microarray Probe Design
AAAAAAAA
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6. Amp and Labeling Experimental Setup
Used Agilent’s Low Input Linear RNA Amplification Kit (LILAK) for Phases II
and III and the Agilent Low Input QuickAmp Kit for Phase IV
Background RNA was Ambion Human Liver Reference and the LIQA reactions
had an input of 10 ng total RNA per reaction for Phase IV
All labeling reactions were performed in triplicate and hybridized as
amp/labeling technical replicates
The Cy3 amp and labeling reactions are doing double duty as One-Color
targets and Two-Color targets
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7. Agilent Microarray Data Analysis
• Concentrated on the “Processed Signals”
• Scanner offset is subtracted
• Background is subtracted
• Multiplicative detrend signal (maybe)
• 75th Percentile scaling across arrays
• Filtered/omitted data from “Non-Uniform” probes
• Wanted to retain the probes that were saturated
• Also wanted to retain the probes were hard to detect
• Analyzed the data for the 10 different ERCC probes to select a single
probe per ERCC transcript for inclusion on every the control grid on
Agilent commercial gene expression microarray format
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8. ERCC Performance in Phase III
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9. Suggested Concentrations of Pools for Modified Latin Square
Experiments (Dilution 1) in ERCC Phase III
Note that concentrations are based on ratios of
the mass of the spike-in RNA transcript to the
mass of background total RNA (based on the
assumptions of an average transcript length of
1000 bases, etc.)
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10. Further Suggested Dilutions for Expanded Range Experiments for
Modified Latin Square Experiments (Dilution 1) in ERCC Phase III
Suggested
Dilution 2 is 1:2
Suggested
Dilution 3 is 1:4
Suggested
Dilution 4 is 1:40
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 We wanted (and needed) higher dilutions for the Agilent microarray platform!

11. Comparing Phase III ERCC Spike-In Dilutions to the Agilent
Spike-In Control Dilutions (mass of ERC per mass of total RNA)
1:12,500
1:37,500
1:125,000
1:375,000
1:1,250,000
1:3,750,000
1:12,500,000
1:125,000,000
1:1,250,000,000
1:12,500,000,000
1:1,000
1:5,000
1:25,000
1:125,000
1:10,000,000
1:50,000,000
1:250,000,000
1:1,250,000,000
1:100,000
1:500,000
1:2,500,000
1:12,500,000
Suggested
Dilutions
100-Fold
Dilutions that
hit Agilent’s
“Sweet Spot”
10,000-Fold
Dilutions that
test the limits of
diluting the
ERCC Spike-Ins
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12. Example of ERCC Performance in Phase III
For the control ERCC-00061
Log2gProcessedSignals
Log2 Mass Spiked
Pools Diluted 1:10,000 Pools Diluted 1:100 Pools Diluted as Described
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13. Can Combine 1-C Data to Test Full Dynamic Range
For the control ERCC-00061
Log2 Mass Spiked
Log2gProcessedSignals
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14. Microarray Data Indicated Some ERCCs Didn’t Dilute As Expected…
ERCC-00073 Was Added to Pool 9 at Wrong Concentration?
Log2gProcessedSignals
Log2 Mass Spiked
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15. Does Distance From 3’ End Make a Difference in Probe Performance?
Distance to the 3’ end of the ERCC on the X-axis
Versus Log2 gProcessed Signals (Or Log10 Ratios) on the Y-axis
Log2gProcessedSignals
Distance (nt) to 3’ End of ERC
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16. Example ERCC Probes That Gave Expected 1-Color Array Signals
For the control ERCC-00028
Pool 10
Pool 9
Pool 8
Pool 7
Liver Control
Log2gProcessedSignals
Distance (nt) to 3’ End of ERC
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 Signals between pools for a given probe
differ 5-fold as expected from the dilutions
Don’t want to select a probe that gives
significant signal in the control sample

17. Example ERCC Probes That Gave Expected 2-Color Array Log10 Ratios
For the same control ERCC-00028
Log10Ratios
Distance (nt) to 3’ End of ERC
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Crosshyb signal from background RNA
results in compressed log ratios …note that
amount of compression observed is sensitive
to amount of ERCC spiked into the mixture
 Good log ratio correlations for microarray
probes located >1000 nt from polyA+ tail

18. ERCC Performance in Phase IV
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19. Construction of the ERCC Phase IV Pools
Each of the 97 ERCC RNA transcripts were diluted to make up 5 different pools
(Pool A through Pool E)
Fewer ERCC spike-in controls were placed at the top end of the dynamic range so
the sample prep reagents would not be swamped by the ERCC spike-in controls
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20. Mixing Sub-Pools at Different Concentrations to Obtain Mixtures 1 to 4
Sub-Pools A thru D were present
at one of 4 concentrations in
each final pool: 40%, 25%,
15%, and 10%
Sub-Pool E was held at a constant
concentration of 10% across all
of the final pools
Comparing ratios across the final
pools test ratios from 1x, 1.5x,
1.6x, 1.7x, 2.5x, 2.7x, and 4x
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21. Agilent Phase IV One-Color “Gummy Worm” Plots
Lower limit of
detection
Saturation
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22. Agilent Phase IV
One-Color Plots
Plus “Gummy” Slope Values
Many of the ERCC
controls have
slopes at or
near 1
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23. Making “Gummy Worm” Plots with Agilent 2-Color Data
 Can you use the
average of the
green and red
signals from the
2-color “self-self”
arrays?
 Yes!
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24. How Do Observed Ratios
Compare to Signal Intensities?
Example: Mix 1 vs. Mix 2
Log2
Ratio
0.68
0.74
0.58
-2.0
0.0 10%
40%
10%
15%
25%
 Log2 Signals are on the X-axis
 Log2 Ratios are on the Y-Axis
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25. Agilent One-Color
Observed Log Ratio
Versus Signal Data
Across All Six Possible
Mixture Combinations
 In general the Agilent
One-Color Microarray
platform does a great job
of measuring ratios that
are very close to the
expected ratios across a
wide dynamic range!
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26. Agilent
One-Color vs.
Two-Color
Performance
 Difference in
saturation levels
likely due to
differences in
the amount of
labeled target
loaded per color
channel
(2x higher for
Agilent One-Color)
One-Color Two-Color
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27. ERCC Performance in Phase V
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28. Example of Agilent Performance in ERCC Phase V Experiments
The Allen Institute for Brain Science has built a “Human Brain Atlas”
which includes gene expression, histological, and MRI data from
several individual post-mortem brains
In phase 1 of this project this project they sectioned the human brains
into ~1,000 parts and analyzed gene expression in each of the 1,000
different areas on Agilent One-Color 8x60K microarrays
The Allen Institute used 20 of the ERCC controls transcripts in a
“barcode” fashion to keep track of samples from a 96-well plate format
The Allen Institute had a control specific for each row and each column of
the 96-well plate to create a specific two ERCC control “barcode” for
each of the 96 possible row-column positions in the plate
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29. Phase V “Barcoding” Experiment Illustrated
1 2 3 4 5 6 7 8 9 10 11 12
A
B
C
D
E
F
G
H
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30. Conclusions
We were able to successfully design 60-mer probes corresponding to each of the
ERCC transcripts and demonstrate robust measurements with IVT labeling
Every Agilent gene expression microarray control grid contains probes to the each
of the 96 ERCC transcripts (replicated 10x on the 8x60K platform)
Agilent and other measurement platforms were sensitive enough to detect
anomalies in the construction of the ERCC test pools
ERCC transcripts diluted in range of 220 demonstrate that microarrays are capable
of detecting gene expression differences spanning over 5 orders of magnitude
The ERCC transcripts help to explore the differences between the one-color and
two-color microarray data
Agilent customers such as the Allen Brain Institute have successfully used the
ERCC controls for sample tracking in their microarray experiments
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31. Thank You!
Agilent:
• Paul Wolber
• Marc Visitacion
• Gary Lin
ERCC 1.0 Phase IV Core Team:
• Marc Salit
• Scott Pine
• Jean Lozach
• Tim Myers
• Sarah Jacob-Helber
• Jenny McDaniel
• Sarah Munro
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32. Stay Tuned Tomorrow…
Joel Myerson (Agilent) Talk Friday
“Efficient Chemical Synthesis of Long and Modified RNA Oligonucleotides”
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33. Back-Up
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34. Visual Effects of ERCC Spike-In Concentrations
Cy5 Pool 10 + Cy3 Pool 8 Liver Control (no ERCs)
Cy5 Pool 10 + Cy3 Pool 10Cy5 Pool 8 + Cy3 Pool 10
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