---------------------------------------------------------------------- {{{ $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 modify it under the terms of the GNU General Public License as published by the Free Software Foundation; either version 3 of the License, or (at your option) any later version. The software is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details. You should have received a copy of the GNU General Public License along with this package. If not, see . }}} ---------------------------------------------------------------------- = 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. 2. Click on the ''Column Chooser'' button to open the column chooser window. 3. 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''. 4. Select displayed subcolumns. The displayed subcolumns should be ''AVG_Signal'', ''BEAD_STDERR'', and ''Avg_NBEADS'', and in this order. 5. 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''. 2. Click on the ''Column Chooser'' button to open the column chooser window. 3. 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''. 4. Select displayed subcolumns. The shown subcolumns should be ''AVG_Signal'', ''BEAD_STDERR'', and ''Avg_NBEADS'', and in this order. 5. 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.