Agilent Feature Extraction 12.2 Reference Guide
Agilent
Feature Extraction 12.2
Reference Guide
For Research Use Only. Not for use in diagnostic
procedures.
Agilent Technologies
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2 Feature Extraction Reference Guide
In This Guide…
This Reference Guide contains tables that list default
parameter values and results for Feature Extraction
analyses, and explanations of how Feature Extraction uses
its algorithms to calculate results.
1 Protocol Default Settings
This chapter includes tables that list the default parameter
values found in the protocols shipped with the software
(Agilent 2- color gene expression (GE), 1-color GE, CGH,
ChIP, miRNA and non- Agilent protocols).
2 QC Report Results
Learn how to read and interpret the QC Reports.
3 Text File Parameters and Results
This chapter contains a listing of parameters and results
within the text file produced after Feature Extraction.
4 XML (MAGE-ML) Results
Refer to this chapter to find the results contained in the
MAGE- ML files generated after Feature Extraction.
5 How Algorithms Calculate Results
Learn how Feature Extraction algorithms calculate the
results that help you interpret your gene expression (2- color
and 1- color), CGH, ChIP and miRNA experiments.
6 Command Line Feature Extraction
This chapter contains the commands and arguments to
integrate Feature Extraction into a completely automated
workflow.
Feature Extraction Reference Guide 3
Acknowledgments
Apache acknowledgment
Part of this software is based on the Xerces XML parser,
Copyright (c) 1999- 2000 The Apache Software Foundation.
All Rights Reserved (www.apache.org).
JPEG acknowledgment
This software is based in part on the work of the
Independent JPEG Group. Copyright (c) 1991- 1998, Thomas
G. Lane. All Rights Reserved.
Loess/Netlib acknowledgment
Part of this software is based on a Loess/Lowess algorithm
and implementation. The authors of Loess/Lowess are
Cleveland, Grosse and Shyu. Copyright (c) 1989, 1992 by
AT&T. Permission to use, copy, modify and distribute this
software for any purpose without fee is hereby granted,
provided that this entire notice in included in all copies of
any software which is or includes a copy or modification of
this software and in all copies of the supporting
documentation for such software.
THIS SOFTWARE IS BEING PROVIDED “AS IS”, WITHOUT
ANY EXPRESS OR IMPLIED WARRANTY. NEITHER THE
AUTHORS NOR AT&T MAKE ANY REPRESENTATION OR
WARRANTY OF ANY KIND CONCERNING THE
MERCHANTABILITY OF THIS SOFTWARE OR ITS FITNESS
FOR ANY PARTICULAR PURPOSE.
Stanford University School of Medicine acknowledgment
Non- Agilent microarray image courtesy of Dr. Roger Wagner,
Division of Cardiovascular Medicine, Stanford University
School of Medicine
Ultimate Grid acknowledgment
This software contains material that is Copyright (c)
1994- 1999 DUNDAS SOFTWARE LTD., All Rights Reserved.
4 Feature Extraction Reference Guide
LibTiff acknowledgement
Part of this software is based upon LibTIFF version 3.8.0.
Copyright (c) 1988- 1997 Sam Leffler
Copyright (c) 1991- 1997 Silicon Graphics, Inc.
Permission to use, copy, modify, distribute, and sell this
software and its documentation for any purpose is hereby
granted without fee, provided that (i) the above copyright
notices and this permission notice appear in all copies of
the software and related documentation, and (ii) the names
of Sam Leffler and Silicon Graphics may not be used in any
advertising or publicity relating to the software without the
specific, prior written permission of Sam Leffler and Silicon
Graphics.
THE SOFTWARE IS PROVIDED “AS- IS” AND WITHOUT
WARRANTY OF ANY KIND, EXPRESS, IMPLIED OR
OTHERWISE, INCLUDING WITHOUT LIMITATION, ANY
WARRANTY OF MERCHANTABILITY OR FITNESS FOR A
PARTICULAR PURPOSE.
IN NO EVENT SHALL SAM LEFFLER OR SILICON
GRAPHICS BE LIABLE FOR ANY SPECIAL, INCIDENTAL,
INDIRECT OR CONSEQUENTIAL DAMAGES OF ANY KIND,
OR ANY DAMAGES WHATSOEVER RESULTING FROM LOSS
OF USE, DATA OR PROFITS, WHETHER OR NOT ADVISED
OF THE POSSIBILITY OF DAMAGE, AND ON ANY THEORY
OF LIABILITY, ARISING OUT OF OR IN CONNECTION WITH
THE USE OR PERFORMANCE OF THIS SOFTWARE.
Feature Extraction Reference Guide 5
6 Feature Extraction Reference Guide
Content
1 Default Protocol Settings 13
Default Protocol Settings—an Introduction 14
Differences between CGH and gene expression microarrays 15
Hidden Settings 15
Tables of Default Protocol Settings 16
CGH_1201_Sep17 16
ChIP_1200_Jun14 24
GE1_1200_Jun14 31
GE2_1200_Dec17 37
GE2-NonAT_1100_Jul11 44
miRNA_1200_Jun14 49
Differences in Protocol Settings Based on Each Step 56
Place Grid 57
Optimize Grid fit 58
Find spots 59
Flag outliers 60
Compute Bkgd, Bias and Error 62
Correct Dye Biases 65
Compute ratios, calculate metrics, and generate results 66
2 QC Report Results 67
QC Reports 68
2-color Gene Expression QC Report 69
1-color Gene Expression QC Report 72
Streamlined CGH QC Report 75
CGH_ChIP QC Report 77
MicroRNA (miRNA) QC Report 79
Non-Agilent GE2 QC Report 81
QC reports with metric sets added 83
Feature Extraction Reference Guide 7
Contents
QC Report Headers 87
2-color Gene Expression QC Report 87
1-color Gene Expression QC Report 88
Streamlined CGH QC Report 88
CGH_ChIP QC Report 88
MicroRNA (miRNA) QC Report 89
Non-Agilent 2-color gene expression QC Report 89
Feature Statistics 90
Spot finding of Four Corners 90
Outlier Stats 91
Spatial Distribution of All Outliers 91
Net Signal Statistics 93
Negative Control Stats 94
Plot of Background-Corrected Signals 95
Histogram of Signals Plot (1-color GE or CGH) 96
Local Background Inliers 97
Foreground Surface Fit 97
Multiplicative Surface Fit 99
Spatial Distribution of Significantly Up-Regulated and Down-Regulated
Features (Positive and Negative Log Ratios) 100
Plot of LogRatio vs. Log ProcessedSignal 101
Spatial Distribution of Median Signals for each Row and Column 102
Histogram of LogRatio plot 103
Inter-Feature Statistics 104
Reproducibility Statistics (%CV Replicated Probes) 104
Microarray Uniformity (2-color only) 106
Sensitivity 107
Reproducibility Plots 108
Spike-in Signal Statistics 111
Spike-in Linearity Check for 2-color Gene Expression 113
Spike-in Linearity Check for 1-color Gene Expression 114
QC Report Results in the FEPARAMS and Stats Tables 121
8 Feature Extraction Reference Guide
QC Metric Set Results 122
CGH_QCMT_Sep17 122
ChIP_QCMT_Jun14 123
GE1_QCMT_Jun14 123
GE2_QCMT_Dec17 124
miRNA_QCMT_Jun14 124
Metric Evaluation Logic 125
3 Text File Parameters and Results 127
Parameters/options (FEPARAMS) 129
FULL FEPARAMS Table 129
COMPACT FEPARAMS Table 151
QC FEPARAMS Table 154
MINIMAL FEPARAMS Table 157
Statistical results (STATS) 160
STATS Table (ALL text output types) 160
Contents
Feature results (FEATURES) 179
FULL Features Table 179
COMPACT Features Table 190
QC Features Table 195
MINIMAL Features Table 201
Other text result file annotations 205
4 MAGE-ML (XML) File Results 207
How Agilent output file formats are used by databases 208
MAGE-ML results 209
Differences between MAGE-ML and text result files 209
Full and Compact Output Packages 209
Tables for Full Output Package 210
Table for Compact Output Package 218
Helpful hints for transferring Agilent output files 222
Feature Extraction Reference Guide 9
Contents
XML output 222
TIFF Results 224
5 How Algorithms Calculate Results 225
Overview of Feature Extraction algorithms 226
Algorithms and functions they perform 226
Algorithms and results they produce 232
XDR Extraction Process 236
What is XDR scanning? 236
XDR Feature Extraction process 236
How the XDR algorithm works 238
Troubleshooting the XDR extraction 239
How each algorithm calculates a result 240
Place Grid 240
Optimize Grid Fit 243
Find Spots 243
Flag Outliers 250
Compute Bkgd, Bias and Error 256
Correct Dye Biases 276
Compute Ratios 280
Calculate Metrics 282
MicroRNA Analysis 285
Example calculations for feature 12519 of Agilent Human 22K image 292
Data from the FEPARAMS table 293
Data from the STATS Table 293
Data from the FEATURES Table 293
6 Command Line Feature Extraction 299
Commands 301
Command line syntax 301
Commands and arguments 302
10 Feature Extraction Reference Guide
Return Codes 307
Extraction Input 309
Extraction Results 314
Status information 314
Examples of status information 315
Error codes from XML file 317
Warning codes from XML file 321
Index 327
Contents
Feature Extraction Reference Guide 11
Contents
12 Feature Extraction Reference Guide
Agilent Feature Extraction 12.2
Reference Guide
1
Default Protocol Settings
Default Protocol Settings—an Introduction 14
Tables of Default Protocol Settings 16
Differences in Protocol Settings Based on Each Step 56
See the Feature Extraction 12.2
User Guide to learn the purpose of
all the parameters and settings and
how to modify them.
Agilent protocols are meant for use
with Agilent microarrays scanned
with an Agilent scanner. They are
intended for use with arrays that
use Agilent default lab procedures
(label, hybridization, wash, and
scanning methods). The
non-Agilent protocol is meant for
use with non-Agilent microarrays
that are scanned with an Agilent
scanner.
When a protocol is assigned to an extraction set, the
software loads a set of protocol parameter values and
settings that affect the process and results for Feature
Extraction.
Parameter values in the protocol depend on the microarray
type and your experiment. The following pages list the
default settings for each of the protocol templates shipped or
downloaded with the software. Each protocol template
represents a different microarray type. You can display these
settings and values when you open the Protocol Editor for
each of the protocol templates.
Agilent Technologies
13
1 Default Protocol Settings
Default Protocol Settings—an Introduction
Default Protocol Settings—an Introduction
To learn more about changing the
default values for the protocols,
see the Feature Extraction 12.2
User Guide.
To learn about the naming of the
protocol templates, see the Feature
Extraction 12.2 User Guide.
Agilent provides new and updated
protocols on the eArray website. If
you set up an eArray login in
Feature Extraction, the software
can automatically download and
install protocol updates from
eArray. See the Feature Extraction
12.2 User Guide for more details.
This chapter presents tables for display of the default
settings for each protocol. Parameter values depend on:
• microarray type
• lab protocol
• formats
• scanner used
Listed in the following table are the names of the
nonremovable protocols and where you can find the tables
that list their default values.
Table 1 Location of protocol template default settings
Protocol Template name Location in chapter
CGH_1201_Sep17
ChIP_1200_Jun14
GE1_1200_Jun14
GE2_1200_Dec17
GE2-NonAT_1100_Jul11
miRNA_1200_Jun14
page 16
page 24
page 31
page 37
page 44
page 49
14 Feature Extraction Reference Guide
Default Protocol Settings 1
CAUTION
Differences between CGH and gene expression microarrays
Differences between CGH and gene expression microarrays
To see the differences in some
default settings between protocols,
go to “GE2_1200_Dec17” on
page 37.
Hidden Settings
CGH microarrays possess a different negative control
sequence scheme than the gene expression microarrays. The
gene expression microarrays have many replicate negative
control features using only one sequence. The CGH
microarrays have many sequences of negative controls that
span the range of sequence variability seen in the biological
probes used on the microarrays. This difference in the
control grid (especially the multiple sequences used for
negative controls) leads to a difference in protocol settings.
To create a protocol for a specific type of microarray, you
are required to use an Agilent- created protocol or
user- created protocol for the same type of microarray.
Protocol templates provide both visible and hidden settings whose
values are specific to the type or format of microarrays. Although you
can change the visible settings so that any two protocols of different
type appear identical, you cannot change the hidden settings that
distinguish these protocols from one another.
Feature Extraction Reference Guide 15
The “Tables of Default Protocol Settings” show only the
default visible parameter values for the steps of the protocol.
You can see the hidden parameters in the FE PARAMS table.
See “Parameters/options (FEPARAMS)” on page 129. Many of
these hidden parameters are image- processing ones that are
chosen using the “Automatically Determine” function.
1 Default Protocol Settings
CAUTION
Tables of Default Protocol Settings
Tables of Default Protocol Settings
These protocol settings may not be optimum for non-Agilent
microarrays or Agilent microarrays processed with non-Agilent
procedures. You determine the settings and values that are optimum
for your system.
CGH_1201_Sep17
This protocol is a CGH protocol for use with the
Oligonucleotide Array- Based CGH for Genomic DNA
Analysis (Enzymatic User Manual version 6.1 or higher, ULS
User Manual version 3.1 or higher).
Table 2 Default settings for CGH_1201_Sep17 protocol
Protocol step Parameter Default Setting/Value (v12.2)
Place Grid Array Format For any format automatically
determined or selected by you, the
software uses the default
Placement Method.
Parameters that apply to specific
formats appear only if that format is
selected.
Placement Method Hidden if Array Format is set to
Enable Background Peak Shifting Hidden if Array Format is set to
Automatically Determine
[Recognized formats: Single
Density (11k, 22k), 25k, Double
Density (44k), 95k, 185k, 185k 10
uM, 65-micron feature size (also
with 10-micron scans), 30-micron
feature size single pack and multi
pack, and Third Party]
Automatically Determine.
Allow Some Distortion (All formats)
Automatically Determine.
Set to False for all arrays except 30
microns single pack and multi pack,
for which it is set to True.
16 Feature Extraction Reference Guide
Default Protocol Settings 1
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Use central part of pack for slope
and skew calculation?
Use the correlation method to
obtain origin X of subgrids
Use Enhanced
Gridding
Optimize Grid Fit Grid Format The parameters and values for
Apply the enhanced gridding
feature released in Feature
Extraction 12.1. The enhancements
include a new iterative method for
determining grid position, rotation,
and skew, and several “fine” grid
tuning methods that improve the
calculation of rotation and skew.
Enhanced gridding also uses both
the foreground and background of
the corner stencil patterns to
improve identification of grid
corners.
optimizing the grid differ depending
on the format.
Hidden if Array Format is set to
Automatically Determine.
Set to False for all arrays except 30
microns single pack and multi pack,
for which it is set to True.
Hidden if Array Format is set to
Automatically Determine.
Set to False for all arrays except 30
microns single pack and multi pack,
for which it is set to True.
True
Note: Results obtained with
protocols that use enhanced
gridding may vary slightly from
results obtained with previous
gridding algorithms (e.g., fewer
gridding errors). Use appropriate
validation processes when
switching from previous CGH
protocols to ones that use
enhanced gridding.
Automatically Determine
[Recognized formats: 65-micron
feature size, 30-micron feature size,
and Third Party]
Iteratively Adjust Corners? Hidden if Array Format is set to
Automatically Determine.
True (All Formats, except Third
Party)
False (Third Party)
Adjustment Threshold Hidden if Array Format is set to
Automatically Determine.
0.300 (All Formats, except Third
Party)
Feature Extraction Reference Guide 17
1 Default Protocol Settings
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Maximum Number of Iterations Hidden if Array Format is set to
Automatically Determine.
5 (All Formats, except Third Party)
Found Spot Threshold Hidden if Array Format is set to
Automatically Determine.
0.200 (All Formats, except Third
Party)
Number of Corner Feature Side
Dimension?
Find Spots Spot Format Depending on the format selected
by the software or by you, the
default settings for this step
change. See the following rows for
the default values for finding spots.
Use the Nominal Diameter from the
Grid Template
Spot Deviation Limit Hidden if Array Format is set to
Calculation of Spot Statistics
Method
Cookie Percentage Hidden if Array Format is set to
Hidden if Array Format is set to
Automatically Determine.
20 (All Formats, except Third Party)
Automatically Determine
[Recognized formats: Single
Density (11k, 22k), 25k, Double
Density (44k), 95k, 185k, 185k 10
uM, 244k 10uM, 65-micron feature
size, 30-micron feature size, and
Third Party]
Hidden if Array Format is set to
Automatically Determine.
True (All Formats)
Automatically Determine.
8.0 for all formats except for third
party, for which it is set to 1.5
Hidden if Array Format is set to
Automatically Determine.
Use Cookie (All Formats)
Automatically Determine.
0.650 (Single Density, 25k)
0.561 (Double Density, 95k)
0.700 (185k, 185k 10 uM, 244k 10
uM, 65-micron feature size)
18 Feature Extraction Reference Guide
Default Protocol Settings 1
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
0.750 (30-micron feature size)
Exclusion Zone Percentage Hidden if Array Format is set to
Automatically Determine.
1.200 (All Formats except 30-micron
feature size)
1.300 (30-micron feature size)
Auto Estimate the Local Radius Hidden if Array Format is set to
Automatically Determine.
True (Single Density, Double
Density, 25k, 95k)
False (185k, 185k 10uM, 65-micron
feature size, 30-micron feature size,
244k 10uM)
LocalBGRadius Hidden if Array Format is set to
Automatically Determine.
100 (when False for 185k, 185k
10uM, 65-micron feature size, 244k
10 uM)
150 (when False for 30-micron
feature size)
Pixel Outlier Rejection Method Inter Quartile Region
(Automatically Determine and All
Formats)
RejectIQRFeat 1.42 (All Formats)
RejectIQRBG 1.42 (All Formats)
Statistical Method for Spot Values from Pixels Use Mean/Standard Deviation
(Automatically Determine and All
Formats)
Feature Extraction Reference Guide 19
1 Default Protocol Settings
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Use Enhanced
SpotFinding
Flag Outliers Compute Population Outliers True
Compute Non Uniform Outliers True
Scanner The values for the parameters
This enhancement allows for more
accurate placement of the center of
each spot by increasing the area
around the expected spot center in
which the algorithm looks for pixels
in the image that are attributable to
that spot. If the increased search
area captures pixels from
neighboring spots, then the
algorithm does not attribute those
pixels to the spot.
Minimum Population 10
IQRatio 1.42
Background IQRatio 1.42
Use Qtest for Small Populations? True
Report Population Outliers as Failed
in MAGEML file
change depending on the scanner
used for the image. See the
following for differences.
False
Note: Results obtained with
protocols that use enhanced spot
finding may vary slightly from
results obtained without spot
finding (e.g., fewer non-uniform
features). Use appropriate
validation processes when
switching to CGH protocols that
use enhanced spot finding.
False
Automatically Determine
Agilent scanner
Automatically Compute OL Polynomial Terms Hidden if Array Format is set to
Automatically Determine.
True
Feature – (%CV)^2 0.04000
Red Poissonian Noise Term
Multiplier
5
20 Feature Extraction Reference Guide
Default Protocol Settings 1
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Compute Bkgd, Bias and
Error
Red Signal Constant Term
Multiplier
Green Poissonian Noise Term
Multiplier
Green Signal Constant Term
Multiplier
Background – (%CV)^2 0.09000
Red Poissonian Noise Term
Multiplier
Red Background Constant Term
Multiplier
Green Poissonian Noise Term
Multiplier
Green Background Constant Term
Multiplier
Background Subtraction Method No Background Subtraction
Significance (for IsPosAndSignif and IsWellAboveBG) Use Error Model for Significance
2-sided t-test of feature vs.
background max p-value
1
5
1
3
1
3
1
0.01
WellAboveMulti 13
Signal Correction—Calculate Surface Fit (required for
Spatial Detrend)
Feature Set for Surface Fit OnlyNegativeControlFeatures
Perform Filtering for Surface Fit False
Perform Spatial Detrending True
Signal Correction—Adjust Background Globally False
Signal Correction—Perform Multiplicative Detrending True
Detrend on Replicates Only False
True
Feature Extraction Reference Guide 21
1 Default Protocol Settings
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Filter Low signal probes from Fit? True
Neg. Ctrl. Threshold Mult. Detrend
Factor
Perform Filtering for Fit Use Window Average
Use polynomial data fit instead of
LOESS?
Polynomial Multiplicative
DetrendDegree
Robust Neg Ctrl Stats? True
Choose universal error, or most conservative Most Conservative
MultErrorGreen 0.1000
MultErrorRed 0.1000
Auto Estimate Add Error Red True
Auto Estimate Add Error Green True
Use Surrogates True
Correct Dye Biases Use Dye Norm List Automatically Determine
Dye Normalization Probe Selection Method Use Rank Consistent Probes
Rank Tolerance 0.050
Variable Rank Tolerance False
3
True
4
Omit Background Population Outliers False
Allow Positive and Negative Controls False
Signal Characteristics OnlyPositiveAndSignificantSignals
Normalization Correction Method Linear
Max Number Ranked Probes -1
Compute Ratios Peg Log Ratio Value 4.00
22 Feature Extraction Reference Guide
Default Protocol Settings 1
CGH_1201_Sep17
Table 2 Default settings for CGH_1201_Sep17 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Calculate Metrics Spikein Target Used False
Min Population for Replicate Stats? 3
Grid Test Format Automatically Determine
Recognized formats: 60 micron and
30 micron feature size, third party
PValue for Differential Expression 0.010000
Percentile Value 75.00
Generate Results Type of QC Report Streamlined CGH
Generate Single Text File True
JPEG Down Sample Factor 4
Feature Extraction Reference Guide 23
1 Default Protocol Settings
ChIP_1200_Jun14
ChIP_1200_Jun14
This protocol is a ChIP protocol for use with Agilent
Mammalian ChIP- on- Chip and DNA methylation applications.
Table 3 Default settings for ChIP_1200_Jun14 protocol
Protocol step Parameter Default Setting/Value (v12.2)
Place Grid Array Format For any format automatically
determined or selected by you, the
software uses the default
Placement Method.
Parameters that apply to specific
formats appear only if that format is
selected.
Placement Method Hidden if Array Format is set to
Enable Background Peak Shifting Hidden if Array Format is set to
Use central part of pack for slope
and skew calculation?
Use the correlation method to
obtain origin X of subgrids
Automatically Determine
[Recognized formats: Single
Density (11k, 22k), 25k, Double
Density (44k), 95k, 185k, 185k 10
uM, 65-micron feature size (also
with 10-micron scans), 30-micron
feature size (single pack and multi
pack) and Third Party]
Automatically Determine.
Allow Some Distortion (All formats)
Automatically Determine.
Set to false for all arrays except 30
microns (single pack and multi
pack), for which it is set to true.
Hidden if Array Format is set to
Automatically Determine.
Set to False for all arrays except 30
microns single pack and multi pack,
for which it is set to True.
Hidden if Array Format is set to
Automatically Determine.
Set to False for all arrays except 30
microns single pack and multi pack,
for which it is set to True.
24 Feature Extraction Reference Guide
Default Protocol Settings 1
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Use Enhanced
Gridding
Optimize Grid Fit Grid Format The parameters and values for
An enhanced automatic gridding
algorithm was released in Feature
Extraction 12.1 for use in CGH
protocols only. Agilent has not
validated the new algorithm in ChIP
protocols.
optimizing the grid differ depending
on the format.
Iteratively Adjust Corners? Hidden if Array Format is set to
Adjustment Threshold Hidden if Array Format is set to
Maximum Number of Iterations Hidden if Array Format is set to
False
Automatically Determine
[Recognized formats: 65-micron
feature size, 30-micron feature size,
and Third Party]
Automatically Determine.
True (All Formats, except Third
Party)
False (Third Party)
Automatically Determine.
0.300(All Formats, except Third
Party)
Automatically Determine.
5 (All Formats, except Third Party)
Found Spot Threshold Hidden if Array Format is set to
Automatically Determine.
0.200 (All Formats, except Third
Party)
Number of Corner Feature Side
Dimension?
Find Spots Spot Format Depending on the format selected
by the software or by you, the
default settings for this step
change. See the following rows for
the default values for finding spots.
Hidden if Array Format is set to
Automatically Determine.
20 (All Formats, except Third Party)
Automatically Determine
[Recognized formats: same as
those listed above except 244k
10uM replaces 65-micron feature
size 10-micron scans]
Feature Extraction Reference Guide 25
1 Default Protocol Settings
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Use the Nominal Diameter from the
Grid Template
Spot Deviation Limit Hidden if Array Format is set to
Calculation of Spot Statistics
Method
Cookie Percentage Hidden if Array Format is set to
Exclusion Zone Percentage Hidden if Array Format is set to
Hidden if Array Format is set to
Automatically Determine.
True (All Formats)
Automatically Determine.
8.0 for all formats except for third
party, for which it is set to 1.5
Hidden if Array Format is set to
Automatically Determine.
Use Cookie (All Formats)
Automatically Determine.
0.650 (Single Density, 25k)
0.561 (Double Density, 95k)
0.700 (185k, 185k 10 uM, 244k 10
uM, 65-micron feature size)
0.750 (30-micron feature size)
Automatically Determine.
1.200 (All Formats except 30-micron
feature size)
1.300 (30-micron feature size)
Auto Estimate the Local Radius Hidden if Array Format is set to
Automatically Determine.
True (Single Density, Double
Density, 25k, 95k)
False (185k, 185k 10uM, 65-micron
feature size, 30-micron feature size,
244k 10uM)
26 Feature Extraction Reference Guide
Default Protocol Settings 1
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
LocalBGRadius Hidden if Array Format is set to
Automatically Determine.
100 (when False for 185k, 185k
10uM, 65-micron feature size, 244k
10 uM)
150 (when False for 30-micron
feature size)
Pixel Outlier Rejection Method Inter Quartile Region
(Automatically Determine and All
Formats)
RejectIQRFeat 1.42 (All Formats)
RejectIQRBG 1.42 (All Formats)
Statistical Method for Spot Values from Pixels Use Mean/Standard Deviation
(Automatically Determine and All
Formats)
Flag Outliers Compute Population Outliers True
Minimum Population 8
IQRatio 1.42
Background IQRatio 1.42
Use Qtest for Small Populations? True
Report Population Outliers as Failed
in MAGEML file
Compute Non Uniform Outliers True
Scanner The values for the parameters
change depending on the scanner
used for the image. See the
following for differences.
Agilent scanner
Automatically Compute OL Polynomial Terms Hidden if Array Format is set to
False
Automatically Determine
Automatically Determine.
True
Feature Extraction Reference Guide 27
1 Default Protocol Settings
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Feature – (%CV)^2 0.04000
Compute Bkgd, Bias and
Error
Red Poissonian Noise Term
Multiplier
Red Signal Constant Term
Multiplier
Green Poissonian Noise Term
Multiplier
Green Signal Constant Term
Multiplier
Background – (%CV)^2 0.09000
Red Poissonian Noise Term
Multiplier
Red Background Constant Term
Multiplier
Green Poissonian Noise Term
Multiplier
Green Background Constant Term
Multiplier
Background Subtraction Method No Background Subtraction
Significance (for IsPosAndSignif and IsWellAboveBG) Use Error Model for Significance
5
1
5
1
3
1
3
1
2-sided t-test of feature vs.
background max p-value
WellAboveMulti 13
Signal Correction—Calculate Surface Fit (required for
Spatial Detrend)
Feature Set for Surface Fit OnlyNegativeControlFeatures
Perform Filtering for Surface Fit False
Perform Spatial Detrending True
0.01
True
28 Feature Extraction Reference Guide
Default Protocol Settings 1
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Signal Correction—Adjust Background Globally False
Signal Correction—Perform Multiplicative Detrending True
Detrend on Replicates Only False
Filter Low signal probes from Fit? True
Neg. Ctrl. Threshold Mult. Detrend
Factor
Perform Filtering for Fit Use Window Average
Use polynomial data fit instead of
LOESS?
Polynomial Multiplicative
DetrendDegree
Robust Neg Ctrl Stats? True
Choose universal error, or most conservative Most Conservative
MultErrorGreen 0.1000
MultErrorRed 0.1000
Auto Estimate Add Error Red True
Auto Estimate Add Error Green True
Use Surrogates True
Correct Dye Biases Use Dye Norm List Automatically Determine
Dye Normalization Probe Selection Method Use Rank Consistent Probes
Rank Tolerance 0.050
Variable Rank Tolerance False
3
True
4
Omit Background Population Outliers False
Allow Positive and Negative Controls False
Signal Characteristics OnlyPositiveAndSignificantSignals
Feature Extraction Reference Guide 29
1 Default Protocol Settings
ChIP_1200_Jun14
Table 3 Default settings for ChIP_1200_Jun14 protocol (continued)
Protocol step Parameter Default Setting/Value (v12.2)
Normalization Correction Method Linear
Max Number Ranked Probes -1
Compute Ratios Peg Log Ratio Value 4.00
Calculate Metrics Spikein Target Used False
Min Population for Replicate Stats? 3
Grid Test Format Automatically Determine
Recognized formats: 60 micron and
30 micron feature size, third party
PValue for Differential Expression 0.010000
Percentile Value 75.00
Generate Results Type of QC Report CGH_ChIP
Generate Single Text File True
JPEG Down Sample Factor 4
30 Feature Extraction Reference Guide
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