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Introduction
The primary use of AltAnalyze is the
calculate statistics to assess alternative splicing,
alternative promoters or other forms of alternative
gene regulation. To do this, AltAnalyze filters
the users raw expression data to remove probe
sets considered to be "not-expressed", calculates
a splicing score (splicing index fold and ttest
p-value), assings exon/intron/splicing annotations
to the high scoring results and further assess
protein, protein domain and microRNA binding site
changes based on these results. AltAnalyze makes
this process relatively easy, with the user only
required to provide three very basic files and
in a simple format. In the following tutorial
we will walk through these steps using a sample
dataset and the results that are subsequently
produced.
Downloading
Sample Data
Before
you run AltAnalyze, you should have either CEL
files or two text files (expression and detection
p-values for all probe sets). AltAnalyze can process
the CEL files you have in order to produce these
two files using builtin calls to the program Affymetrix
Power Tools. To download sample CEL files, click
here. Otherwise, you can download already
processed AltAnalyze expression files from here.
These files have data for all 1.4 million probesets
on the human Exon 1.0 array.
Installing AltAnalyze and Saving Your Data
AltAnalyze
can be downloaded for multiple operating systems
from http://AltAnalyze.org.
Once you have downloaded the compressed archives
to your computer, extract these to an accessible
folder on hard-drive (e.g., your user account).
Creating
a Comparison and Groups File (OPTIONAL)
If
your dataset has over 30 CEL files or dozens of
groups, it may save you time to make the groups
and comps files in advance. Although not recommended
when working with this sample dataset, go
here if this applies to your own dataset.
Running
AltAnalyze
Now you are ready to process
your raw input files and obtain alternative probe
sets with splicing and functional annotations.
To proceed:
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1)
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Open
the AltAnalyze_v1beta folder and select the
binary file "AltAnalyze". In Windows, this
file has the extension ".exe". If you are
working on a Linux machine or are having problems
starting AltAnalyze, you can also start the
program directly from the source
code. |
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2) |
(AltAnalyze:
Introduction) In the resulting introduction
window, select "Begin Analysis". |
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3) |
(AltAnalyze:
Main Dataset Parameters) Select the species
type "Homo sapiens" and "Continue". |
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4) |
(AltAnalyze:
Main Dataset Parameters) Select the "CEL files"
button and the array type "Affymetrix Exon
ST 1.0 Array". |
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5) |
(AltAnalyze:
Select CEL files for APT) For dataset name,
type in "hESC_differentiation", select the
directory containing the CEL files (make sure
these have been extracted from the TAR file
and Gzip files) and then select an empty directory
to save the results to. |
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6) |
(AltAnalyze:
Select CEL files for APT) In the resulting
warning window, select "download"
for AltAnalyze to automatically download and
install the library and annotaiton files for
that array. |
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7) |
(AltAnalyze:
Expression Analysis Parameters) Accept the
default parameters by clicking "Continue".
These coptions can be modified later on, if
you wish to increase or decrease the stringency
of the analysis. |
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8) |
(AltAnalyze:
Alternative Exon Analysis Parameters) Accept
the default parameters by clicking "Continue".
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9) |
(AltAnalyze:
Assign CEL files to a Group) Type in the group
name for each sample (first three are hESC
and second three are cardiac_precursors).
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10) |
(AltAnalyze:
Establish All Pairwise Comparisons) Selects
the groups to compare (cardiac_precursors
vs. hESC). |
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11) |
A
new window will appear that displays the progress
of the analysis. Analysis of the sample dataset
should take approximately 20 minutes, but
can take longer depending upon the machine
and operating system. When finished, AltAnalyze
will display a new pop-up window, informing
the user that the analysis is complete. If
analyzing multiple experimental groups, these
will be run in succession. |
Interpreting
the Results
Proceed
here.
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