Microarray image processing must correctly identify the regular grid of features within an image and independently quantify the fluorescence intensity for each feature. Image artefacts must be additionally identified and removed from the overall analysis. Fluorescence intensities directly indicate the abundance of each sequence, since the sequence of each probe on the array is already known.
The first steps of RNA-seq also include similar image processing; however, conversion of images to sequence data is typically handled automatically by the instrument software. The Illumina sequencing-by-synthesis method results in an array of clusters distributed over the surface of a flow cell. The flow cell is imaged up to four times during each sequencing cycle, with tens to hundreds of cycles in total. Flow cell clusters are analogous to microarray spots and must be correctly identified during the early stages of the sequencing process. In Roche’s pyrosequencing method, the intensity of emitted light determines the number of consecutive nucleotides in a homopolymer repeat. There are many variants on these methods, each with a different error profile for the resulting data.