Vizxlabs GENESIFTER 2005 User Manual

User’s Guide

GeneSifter Overview

• Login

• Upload Tools

• Pairwise Analysis

• Create Projects

For more information
about a feature see the
corresponding page in the
User’s Guide noted in the
blue circle ( ) .

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Upload Tools

Upload microarray data files.

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Scatter Plot

Interactive scatter plot provides
visualization of the entire array
data set and identification of
individual genes.

Ontology Report

Summarize ontology terms
for a gene list and assess
the biological significance
of the genes within the list.

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User Login

Access your secure
account from any
computer (PC or Mac)
with Internet access.

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Create New Project

Create user-defined projects
with two or more groups. See
next page for project analysis
options.

Pairwise Analysis

Define two groups and apply
normalization, statistical
analysis and quality metrics to
create lists of differentially
expressed genes.

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One-Click Gene Summary™

Provides a synopsis of the most
current information available for the
genes on your array. It includes
information from UniGene,
LocusLink, Gene Ontology terms
and more.

Export Results

Export data and gene
annotation to Excel.

GeneSifter Overview

• Project Analysis

• Filtering

• Function Navigation

• Pattern Navigation

•Clustering

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Filtering

Apply fold
change cutoffs,
statistical
analysis and
quality metrics

to create lists of
differentially
expressed
genes.

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Clustering

Identify patterns of gene expression
with unsupervised clustering
functions.

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Ontology Report

Summarize ontology terms
for a gene list and assess
the biological significance
of the genes within the list.

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*

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Project Analysis

Project analysis functions
allow analysis across all
conditions in a project.

Pattern Navigation

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Define and identify patterns of gene
expression with supervised clustering.

Function Navigation

Rapidly identify and group genes based on
function using Gene Ontology terms.

Ontology Report, Cluster

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Samples and the One-Click
Gene Summary are
available for all types of
project analysis.

Cluster Samples

Use hierarchical clustering to
determine the relationship of
samples based on a gene list.

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One-Click Gene Summary™

Includes information from UniGene,
LocusLink, Gene Ontology terms

and more.

GeneSifter

Introduction and Login

Welcome to GeneSifter, the web­based microarray data management
and analysis system, which relies on
VizX Labs’ BIOME™ bioinformatics
software engine. This document gives
an overview of some of the features
available in Genesifter. To get started
from www.genesifter.net
these steps:

1. Select the Login button from the

top right corner.

2. Enter your user name and
password in their respective
prompts and click on the Login
button.

4. A successful login should show a
screen with control panel on the left
and the most recent
announcements concerning
Genesifter.

please follow

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Genesifter Support and Sales

E-mail: [e-mail]
Toll-free: 1-877-WEB-GENE
Direct: 206-283-4363

Genesifter

Online Help

Genesifter provides page-specific
online help.

1. Click on the help icon ( ) to
access page-specific help
documents. The help icon can be
found in the upper right corner of
most pages.

2. Clicking on the help icon will
open a new browser window
which will list the help available
for that particular page. Select
the document you wish to view.

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Uploading Data

Upload Tools

1. In order to upload data, select

Upload Tools from the control
panel on the left.

2. GeneSifter offers four tools to
load data. The application you
use will depend on the format
and origins of the data being
loaded. QuickLoad Wizard,

Batch Upload, FlexLoad
Wizard and Advanced Upload
Methods are further described

in the following pages.

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Uploading Data

Using QuickLoad Wizard

Use the QuickLoad Wizard to load your
data into GeneSifter. Supported
platforms for this tool include:

• Affymetrix (native CHP files, or CHP
files saved as tab-delimited text)

• Codelink

• Pathways™ 2 & 4

• Spot-On

• Mergen arrays scanned with
GenePix®

(all other GenePix files may be loaded
using FlexLoad)

Data Files may be archived (zipped) prior
to upload.

1. Select Upload Tools from the

control panel on the left.

2. Select Run QuickLoad Wizard from

the Upload Tools page. A new
window will guide the user in the
upload process.

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Uploading Data

Using QuickLoad Wizard (continued)

3. Select the array manufacturer or the image
analysis software used and then click the
Next button.

4. Select your array from the list of available
arrays. If your array is not listed, please

contact scientific support for
information on adding the array.After
you have selected your array, click Next.

Note for Affymetrix Users: Auto Column

Detection should work for data from MAS 5
and GCOS.

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Uploading Data

Using QuickLoad Wizard (continued)

5. Now you will enter information about

the sample (referred to as “Target”)
that was hybridized to the array. If
you have already entered
information about the target, select it
from the Select Target pull-down
menu, otherwise enter your target
using Create New Target.

6. If you create a new target, you will
need to select an appropriate
“Condition” from the pull-down
menu. Otherwise, enter your
condition using Create New
Condition.

7. Select Next when you have entered
all needed information.

8. If your array has two channels,
repeat steps 5, 6, and 7 for the
second channel.

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Uploading Data

Using QuickLoad Wizard (continued)

9. Select Browse and find your data file
on your local computer. Select the file
and then click the Next button to
upload the file to GeneSifter.

10. You will now see a summary of the
information you have provided. You
can enter a description for the
experiment(s) being uploaded. Select
Save Data to save the data in your
GeneSifter account.

11. When the data is successfully
uploaded, you will see Success! as the
Last Upload status. Common reasons
for failure include: data not in the
correct format, or selecting the wrong
array at step 4.

12. After saving, you can either load more
data by selecting Next or exit the

QuickLoad Wizard by selecting Done.

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Uploading Data

Using QuickLoad Wizard

Affymetrix Manual Column Detection

The Upload Wizard will automatically detect
the proper data columns for text files
generated from MAS 4, MAS 5 and GCOS. If
auto detection fails, you can use Manual
Column Detection.

1. Affymetrix MAS 5 data format. This is
an example of a file from the U34B
GeneChip®. Formats may vary due to
differences in export from MAS 5.
Generally the first column will contain
the probeset ID. This identifies each
gene on the array. The two columns
that are needed by GeneSifter for
analysis are the columns containing the
signal and detection value for that
probe set. In this example, column B,
which is labeled Signal, contains the
derived signal value and column C,
labeled Detection, contains the quality
value. There may be additional
columns present in a data file and the
heading for the signal and quality
values may be different than what is
presented here. In general the signal
column will either be labeled Signal or
will have the word Signal at the end of
the column name. This column can
contain both positive and negative
numbers. The quality column will
generally be labeled Detection or will
have the word Detection at the end of
the name. This column will contain the
letters A, M and P.

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Uploading Data

Using QuickLoad Wizard

Affymetrix Manual Column Detection

(continued)

2. Select Run QuickLoad Wizard as
usual (see preceding description).
Select Manual from pull-down menu.

3. Enter information about columns in the
data file. In the sample file the
probeset ID is in the first column
(column A), the signal is in column B
and the detection call value is in
column C, so A would be entered for
Probeset Column, B would be entered
for Signal Column and C would be
entered for Detection Call Column. In
the sample file the data begins on the
second line of the file so 2 is entered
for Data starts on line.

4. Select Next and continue as usual for
QuickLoad Wizard. The setting will
be saved and used to correctly upload
the data.

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Uploading Data

Using Batch Upload

Use Batch Upload to load multiple data
sets stored in a spreadsheet as a tab­delimited text file. Note: See step 7 for a
description of required file format.

1. Select Upload Tools from the
control panel on the left.

2. Select Run Batch Upload.

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Uploading Data

Using Batch Upload (continued)

3. Enter a name and description for
the array you are uploading. The
pull-down menu has options for
the type of data being loaded
including:

Use Affymetrix Probeset IDs –
use if the first column of your file
contains Affymetrix Probeset IDs
instead of GenBank accession
numbers.

This File is a GEO Data Set –
use if you downloaded data from
GEO as a GEO dataset.

Use CodeLink Quality Values –

use if your file has the CodeLink
flags G, M, L.

4. Browse your computer to find the
file containing your data. Use the
Select Array pull-down if you
have previously loaded data from
this array and you wish to add to
that data set.

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5. Select Upload Data.

6. Data uploaded. You can either
exit Batch Upload by selecting

Done or select Upload More
Data.

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Uploading Data

Using Batch Upload (continued)

7. The file to be loaded must be a tab­delimited text file (txt). GeneSifter
does not accept Excel spreadsheets
(xls).

The first column
figure) should contain an identifier
for that gene. Accepted identifiers
are:

 Accession Number
 IMAGE Clone ID
 Affymetrix Probeset IDs

The second column (Column B) is
for an internal identifier. This is left
to the discretion of the user and can
be left blank.

The next 2 columns contain the
intensity (Column C) and quality
values (Column D) for that gene in
the first experiment to be loaded.
Additional experiments are added in
the same way (one column for
intensity, one for quality).

(Column A in the

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The first row
A: This cell can be empty.
B: This cell can be empty.
C: Target name for Experiment #1.
D: Condition for Experiment #1.
E: Target name for Experiment #2.
F: Condition name for Experiment #2,

etc.

must contain this information.

Uploading Data

Using Batch Upload (continued)

8. File format for Batch Upload using

housekeeping genes. The format is
the same with one exception: the
third column must be labeled HKG
(housekeeping genes). The genes
that are designated as
housekeeping should be marked
with an x in this column. The
intensities and quality values should
follow as stated for Batch Upload
without housekeeping genes.

In this example the genes in rows 7 and 9
(TRIM9 and GOLGB1) have been
designated as housekeeping genes.

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Uploading Data

Using FlexLoad Wizard

Use the FlexLoad Wizard to load data in
GeneSifter if the array you are using is
not included in the QuickLoad Wizard.
Familiarity with the layout of your files is
advised before going any further. You will
be asked:

• to provide information about the file
structure, e.g. what column
describes absolute intensity,
background intensity, etc.

• how you want the data transformed,
e.g. preserve channel intensities or
express as a ratio of the two
channels.

• how you want the data normalized,
e.g. LOWESS

1. Select Upload Tools from the

control panel on the left.

2. Select Run FlexLoad Wizard.

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Uploading Data

Using FlexLoad Wizard (continued)

1. The Protocol Title is the name

given to a protocol (i.e. the settings
for a specific type of file to be
uploaded). You can select a
protocol you have already
generated or create a new one.

2. If creating a new protocol, replace

“Untitled Protocol” with a Protocol
Title.

3. Enter an optional Description for

the protocol.

4. Click on Create New to begin the

creation of a new protocol.

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Uploading Data

Using FlexLoad Wizard (continued)

5. If you previously loaded the array
into your account, select it from the
menu list. Alternatively, if you are
creating a new protocol, enter the
name of the array in the Create New
Array field.

6. Select the number of Channels.

7. Enter the number of files you will be
uploading (the maximum allowed at
any one time is 30).

8. If you know that the genes are all
listed in the same order in every file
(experiment) then select Same
Order.

If you select Unique IDs, FlexLoad
will not assume identical gene order
in each file, but instead will utilize a
supplied unique identifier. If Unique
IDs is selected, every ID for that
array must be unique and there
cannot be any blank data rows.
Ideally, you should use a GenBank
Accession Number or an Image
Clone ID as the Unique ID to assist
in populating the One-Click Gene
Summary.

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Uploading Data

Using FlexLoad Wizard (continued)

If your data has two channels, from Step 6:

9. Select how you want your data

represented:

Intensities

Data for each channel will be stored
separately in GeneSifter.

Ratios

Generates a ratio of the intensities of
the red and green channels.
GeneSifter only saves the ratio to your
account.

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Uploading Data

Using FlexLoad Wizard (continued)

10. Provide the column number or letter
that contains the gene ID.

11. Identify the type of gene ID by
selecting either Auto Detect,

Accession Number, IMAGE Clone
ID, or Other. If Accession Number

or IMAGE Clone ID is selected and
the data file contains other identifiers
or blank rows, errors may occur. In
general Auto Detect should be
used.

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12. Optionally, indicate the column
number for gene annotation, if
available.

13. Indicate the row number where the
intensity data begins. Do not include
the column headings.

14. Provide the column numbers that
contain the respective data. For
example, if your data file contains
the cy3 intensity in column 8 and
column 10 contains the background,
enter 8 for Col 1 and 10 for Col 2 to
specify these values for the cy-3
intensities.

Op refers to the operation to perform
between the two values. Op may be
used to subtract background, or take
the ratio of foreground/background
for quality control purposes. It is not
necessary to enter any value for Op,
or Col 2.

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Uploading Data

Using FlexLoad Wizard (continued)

This window appears only if
you chose Ratios in Step 9.

15. Perform LOWESS Normalization
on the data.

16. Select how you want to normalize
the data.

17. Method for calculating the ratio of
intensities. Per file basis allows
you to take into account any dye-

swap experiments you may have.

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Uploading Data

Using FlexLoad Wizard (continued)

18. If your targets already exist, you
can select them from the pull­down menu. If you need to
create new targets, use
Advanced Settings.

19. Select Browse to upload the
file(s). If you were uploading
more files, additional rows would
be present. In this screen, two
files are being uploaded.

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20. For two-color arrays, if Per file
basis was selected in step 17,

indicate whether you want the
ratios to be cy5/cy3 (5/3) or
cy3/cy5 (3/5).

21. A summary of parameters that
have been previously selected
can be viewed.

22. Select Advanced Settings if
you need to enter the target and
condition information, or to
change the number of files being
loaded.

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Uploading Data

Using FlexLoad Wizard (continued)

23. Upon selecting Advanced
Settings, you can change the

number of files to be loaded.

24. You also have the ability to “Create
New Targets/Conditions”. Enter
target and condition information for
each file to be loaded in the top
portion of the screen. Otherwise,
select “Use Pre-existing Targets”.

25. You can save the output as a text
file formatted for Batch Upload by
selecting Save as File or load it
directly into GeneSifter by selecting

Upload Files

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Uploading Data

Advanced Upload Methods

Robust multi-array average (RMA) is a
method for deriving expression
measurements from the probe level data
contained in an Affymetrix CEL file.

GeneSifter users have the option to
perform RMA or GC-RMA during the
upload of CEL files. The normalized data
is saved in the user’s account for further
analysis.

1. Locate the Affymetrix .CEL files
you want to load on your local
computer.

2. All the files to be uploaded and
transformed using RMA need to
be compressed into a single ZIP
file for upload.

3. Select Upload Tools from the
Control Panel.

4. Click on Run Advanced

Upload Methods to begin.

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Uploading Data

Advanced Upload Methods

(continued)

5. Select the normalization
method. Select the type of
Affymetrix array used in your
experiments. If your array is
not listed, please contact
scientific support
([e-mail]
help loading your array format.

6. Click the Next button to
continue.

7. Click Browse to locate the .zip
archive containing the CEL files
on your local disk. Please note
that all the files to be loaded
need to be contained within a
single .zip file.

8. Choose how you want to create
targets and conditions for the
loaded files.

9. Click Next to continue.

) for

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