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Last change on this file was 3477, checked in by Jari Häkkinen, 6 years ago

Added note on what is considered a negative control.

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$Id: README_PluginDetails 3477 2015-09-03 12:01:58Z jari $

Introduction

There are many plug-ins in the Illumina plug-in package for BASE. This file gives detailed information on some of the contributed plug-ins.

Illumina expression background correction plug-in

This plug-in will remove a per-slide global background from all spots. The background is calculated from a set of negative control spot on the array. See implementation details below for details.

Parameters

There is one parameter to set that specifies how background intensities should be calculated. Allowed values are median or mean, i.e. the background is either the median or the mean of the negative control spots on the array.

The expression of the background probes is optionally saved to a file. The default is not to save the expression matrix but this can be changed during job configuration.

Implementation details

Each assay is treated separately, i.e., no samples are combined together. All calculations are made on the current bioassay data implying that this plug-in should be used early in analysis and before background spots are removed.

A spot is considered to be a negative control spot if it has a Control group name exactly matching the string negative.

Illumina detection P-value calculation

This plug-in implements BeadStudio like detection P-value calculations for Illumina expression data (see http://www.genomecenter.ucdavis.edu/expression_analysis/documents/illumina_normalization_081201.pdf) on detection P-values.

The plug-in will always base the detection P-value calculation on raw data values, i.e., the mean raw intensity for the different signals. By default the calculations are based on negative controls available in the root bioassay set for the current analysis branch. The user may change this to only use negative controls in the current bioassay set.

The detection P-value plug-in does not filter the assays, it provides the detection P-values usable in a filter step after running this plug-in.

Parameters

The plug-in requires input of array type since detection P-value are calculated differently depending on array type.

Users may select to use negative controls in the current bioassay set only. The default behaviour is to use all negative controls in the root bioassay set for the current bioassay set.

A cut off parameter is available to exclude outliers within the negative controls. The cutoff defines the acceptable negative control signal range

median-MAD*cutoff < I < median+MAD*cutoff

where MAD is the median absolute deviation.

Implementation details

Each assay is treated separately, i.e., no samples are combined together. All calculations are made on the raw bead-type level data, i.e., on the average expression value for each bead type and raw data is always used irrespective when in analysis the detection P-value is calculated.

Pvalue calculation for whole genome arrays:

For all signals i calculate the detection P-value as Pvalue = 1-R/N where R is the rank of the signal i relative to the negative controls and N is the number of negative controls.

Pvalue calculation for others array types (DASL, miRNA, VeraCode DASL, and Focused Arrays):

For all signals i calculate the detection P-value as Pvalue = 1/2 - 1/2 * erf( [i-AvgControl]/StdControl/sqrt(2) ) where AvgControl is the average intensity of the negative controls, StdControl is the standard deviation of the the negative controls, and erf is the error function (http://mathworld.wolfram.com/Erf.html). The error function is used for arguments within the range (-4,4). To save CPU cycles, the function value for arguments outside this range is set to -1 and 1, respectively.

A spot is considered to be a negative control spot if it has an Control group name exactly matching the string negative.

Control Summary plots

This extension provides overview plots for Illumina expression data. The 'Overview plots' tab becomes available in the experiment analysis tree when the user selects a bioassay set. The plots are automatically generated when the user clicks on the Overview plots tab. The display of the plots cannot be changed by the user and the same plot is shown irrespective which bioassay set is selected.

Currently two control summary curves are generated in one plot; The average intensity of perfect match beads in each assay, and the average intensity of housekeeping beads in each assay.

The average intensity I_avg is calculated as

I_avg = sum[Iraw_i] / N

where N is the number of bead types in the sum, Iraw_i is the raw mean intensity for bead type i.

A bead type is considered to belong to the perfect match group if it is annotated with ':pm' in the reporter annotation column '[Rep] Control group id', and a bead type is grouped as housekeeping if it is annotated with 'housekeeping' in reporter annotator column '[Rep] Control group id'.


Copyright (C) 2009, 2010 Jari Häkkinen
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 BASE. If not, see <http://www.gnu.org/licenses/>.

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