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Before microarray data can be analysed it is necessary to perform a series of preprocessing steps.
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Background substraction Chip-wise rescaling: These genesprihg the “raw” data we will use for the exercise. In this part of the tutorial we will use R to repeat the operations Berry et al.
You will note that each chip has a mean value benespring This is due to the sample-wise centring applied by Berry using the BeadStudio software. Furthermore, in each chip the mean differs from the median. This is symptomatic of asymmetrical distributions.
A solution to this problem is to set the small values to an arbitrarily defined threshold. In Berry et al. Note however that 1, spots out of 2, have a value lower than 10, what means that more than two thirds of the values are changed before any analysis is performed.
This has a consequence that all the subsequent gene-wise estimators mean, median will be strongly biased. For example, for many genes all values will be set to 10, so that variance filtering is not relevant anymore.
The histogram with the raw intensitiies for the 42 samples in the range In R we can do this by applying a median calculation over all rows of the data frame training. Then we can simply divide the data frame by the vector ; the operation is automatically repeated for each column.
Note that the result of this operation geneespring that all the expression values are set to small numbers and the chip medians are set to 1. We loose information about which genes are strongly expressed and which are weakly expressed, we only keep information about the relative change in expression for different genes.
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A gene is considered present if its Illumina detection call p-value is smaller than 0. Since there can be spurious signals caused by dust, etc.
The histogram with the number of present samples per gene Note how there are more than 27, genes ‘present’ in not a single chip, more than genes ‘present’ in all 42 chips and lesser numbers anywhere in between. The red bar at position 4 4.
The second filter removes genes that are not expressed differentially. A gene is considered to be significantly expressed downward or upward is there is at least a twofold change relative to the reference.
Since there can be spurious outliers due to dust, etc. Note that we do not have to re-compute the median since we know that it is 1.