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

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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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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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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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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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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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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ERCC Performance in Phase III

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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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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!

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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Example of ERCC Performance in Phase III For the control ERCC-00061

Lo

g2 g

Pro

ce

sse

d S

ign

als

Log2 Mass Spiked

Pools Diluted 1:10,000 Pools Diluted 1:100 Pools Diluted as Described

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Can Combine 1-C Data to Test Full Dynamic Range For the control ERCC-00061

Log2 Mass Spiked

Log

2 g

Pro

cessed S

ignals

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Microarray Data Indicated Some ERCCs Didn’t Dilute As Expected… ERCC-00073 Was Added to Pool 9 at Wrong Concentration?

Lo

g2 g

Pro

ce

sse

d S

ign

als

Log2 Mass Spiked

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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

Lo

g2 g

Pro

ce

sse

d S

ign

als

Distance (nt) to 3’ End of ERC 7/10/2014

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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

Lo

g2 g

Pro

ce

sse

d S

ign

als

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

Example ERCC Probes That Gave Expected 2-Color Array Log10 Ratios For the same control ERCC-00028

Lo

g1

0 R

atio

s

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

ERCC Performance in Phase IV

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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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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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Agilent Phase IV One-Color “Gummy Worm” Plots

Lower limit of detection

Saturation

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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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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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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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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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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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ERCC Performance in Phase V

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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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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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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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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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Stay Tuned Tomorrow…

Joel Myerson (Agilent) Talk Friday

“Efficient Chemical Synthesis of Long and Modified RNA Oligonucleotides”

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Back-Up

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Visual Effects of ERCC Spike-In Concentrations

Cy5 Pool 10 + Cy3 Pool 8 Liver Control (no ERCs)

Cy5 Pool 10 + Cy3 Pool 10 Cy5 Pool 8 + Cy3 Pool 10

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