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$Id: README_beadstudio2ibs.txt 940 2009-01-27 13:37:03Z martin $
Copyright (C) 2009 Markus Ringnér
This file is part of Illumina plug-in package for BASE.
Available at http://baseplugins.thep.lu.se/
BASE main site: http://base.thep.lu.se/
This is free software; you can redistribute it and/or
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MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE.  See the
GNU General Public License for more details.
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Importing Illumina data from BeadStudio into BASE

This file contains information about how to generate expression data for the Illumina plug-in package from BeadStudio.

Background

For expression data, the Illumina plug-ins package for BASE requires Illumina Bead Summary (IBS) files. IBS files contain bead-type level data for scanned Sentrix arrays. IBS files are simple comma separated text files with file extension .csv. IBS files are not outputted by a BeadArray Reader with default settings, but a local Illumina Field Application Scientist can configure the BeadArray Reader to output IBS files. This document describes how to generate IBS files from BeadStudio without requiring any changes of the default settings for the BeadArray Reader.

BeadStudio

Create BeadStudio project

Generate a BeadStudio gene expression project with the experiments you want to generate IBS files for. The IBS files should contain data that is neither normalized nor background subtracted so:

  • Analysis Type should be set to Gene Expression.
  • Set the parameter Normalization to none.
  • The parameter button Subtract Background should be unchecked.

Create and export BeadStudio tables

Two tables should be exported: Sample Probe Profile and Control Probe Profile.

Note that the tables must be exactly as specified here: no additional columns are allowed and the order of the columns needs to be as specified.

Sample Probe Profile

  1. Click on the Sample Probe Profile tab to view the table.
  1. Click on the Column Chooser button to open the column chooser window.
  1. Select displayed columns. The first column should be PROBE_ID, followed by one column per sample, for example, 4313143087_A, 4313143087_B, ...., and the last column should be ProbeID.
  1. Select displayed subcolumns. The displayed subcolumns should be AVG_Signal, BEAD_STDERR, and Avg_NBEADS, and in this order.
  1. Export the table by clicking on the Export displayed data to a file button. Save as type Tab delimited (*.txt). In the following it is assumed that you have named the file sample_probe_profile.txt.

Control Probe Profile

  1. Click on the Control Probe Profile tab to view the table. If there is no Control Probe Profile tab, then first enable it under the pull-down menu Window.
  1. Click on the Column Chooser button to open the column chooser window.
  1. Select displayed columns. The first displayed column should be ProbeID, followed by one column per sample, for example, 4313143087_A, 4313143087_B, .... The selection of samples should be the same and in the same order as in the exported Sample Probe Profile.
  1. Select displayed subcolumns. The shown subcolumns should be AVG_Signal, BEAD_STDERR, and Avg_NBEADS, and in this order.
  1. Export the table by clicking on the Export displayed data to a file button. Save as type Tab delimited (*.txt). In the following it is assumed that you have named the file control_probe_profile.txt.

Convert BeadStudio tables to IBS files

The two files exported from BeadStudio with data for the sample probes and the control probes can be converted into an IBS file for each sample using beadstudio2ibs.pl with the following command:

perl beadstudio2ibs.pl --samples_file=sample_probe_profile.txt --controls_file=control_probe_profile.txt

Import IBS files generated from BeadStudio into BASE

The IBS files generated by beadstudio2ibs.pl can be imported into BASE using the following settings:

  • Use the raw data type for the Illumina plug-in package: Illumina Bead Summary (IBS)
  • Use the option expression 1.

Additional remarks

  • The choice between expression 1 and expression 2 (and possibly more alternatives in the future) should be made to match the number of IBS files per sample. When IBS files are outputted directly from the BeadArray Reader one .csv file is produced per strip on the array. BeadStudio does not keep track of strips and therefore a single .csv file per sample is generated by beadstudio2ibs.pl, regardless of the number of strips on the beadchips used. This means, for example, that for HumanWG-6 v3.0 beadchips (two strips per sample) expression 2 should be used when IBS files are generated from the scanner and expression 1 should be used when IBS file are generated from BeadStudio data.
  • The data in an IBS file from BeadStudio will not be identical to the data in an IBS file from the scanner. The reason for this difference is that the scanner outputs the average intensity for all beads, whereas BeadStudio removes outlier beads in its calculation of averages.
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