Standard Illumina Image Processing}

The first step in a pipeline for image processing is to read both the TIFF image and the bead-level text file. The text file contains the identities of each bead, as well as the bead-centre coordinates. The image processing methods contained within beadarray use these coordinates as seed points for intensity extraction, although one can conceive of approaches where bead-centres are calculated seperately, prior to intensity extraction. However, even with such an approach the Probe ID for each bead will need to be extracted from the .txt file.

tiff <- readTIFF()
data <- readBeadLevelTextFile()

The standard method employed by Illumina’s scanner for calculating bead intensity is a four step process, described in Kuhn et al . It can be summarized as:

• Calculate background value
• Sharpen image
• Calculate foreground value
• Subtract background for foreground to give final intensity

If the function readIllumina() is called with useImages = TRUE then intensities are extracted using code that gives a very close emulation of that used by Illumina. If one wished to perform this calculation themselves (outside of readIllumina(), it can be done using the following code.

bg <- illuminaBackground(tiff, data[,3:4])
tiffSharp <- illuminaSharpen(tiff)
fg <- illuminaForeground(tiffSharp, data[,3:4])
finalIntensity <- fg - bg

Each of the functions above take a matrix representing the pixel values from the TIFF image as their first argument. The background and foreground algorithms additionally take a two column matrix containing the coordinates of the bead centres. If one wished to calculate intensities for only a subset of the beads then supplying only the appropriate bead-centres in this step would achieve this

After calculating intensities they need to be inserted into a beadLevelData object. The code below shows how to create a new object and insert intensity values. However, this approach creates an empty beadLevelData object, which will be lacking any information except that which the user manually inserts.

BLData <- new(Class = "beadLevelData")
BLData <- insertBeadData(BLData, array = 1, what = "Grn", data = finalIntensity)

An easier alternative to creating your own beadLevelData object is to use the function readIllumina() to read the data as described in the main vignette. This ensures that any available data (such as sample IDs, scanner metrics, grid sizes etc.) are read in and stored. One can then choose to overwrite the values generated by readIllumina(), or store alternative intensities alongside them.

The example below first reads the data using the standard arguments to readIllumina(), which will extract the intensities from the .txt file. The second step overwrites those intensities with those we calculated previously (which should be very similar). The final command creates a new entry in the beadLevelData object (refered to as GrnLog, that stores the log transform of the values we calculated earlier. In this way the user can store a variety of intensity values if they wish to experiment with alternative forms of background subtraction, gradient removal etc.

BLData <- readIllumina()
BLData <- insertBeadData(BLData, array = 1, what = "Grn", data = finalIntensity)
BLData <- insertBeadData(BLData, array = 1, what = "GrnLog", data = log2(finalIntensity))