Editing BioMicroCenter:IlluminaDataPipeline (section)

=== Base Calling ===
''Firecrest'' is the module used for image analysis. Firecrest identifies cluster positions and extracts intensities. Through image filtering, it
sharpens and enhances clusters, removes background noise, and detects clusters based on morphological features on the image. Firecrest also adjusts the scale and registration of an image. Firecrest is currently performed in real time with the sequencing process on a dedicated IPARR server as part of the 1.0 pipeline.

''Bustard'' is the module used for base calling. Bustard deconvolves the signal from the clusters and applies correction for cross-talk, phasing, and prephasing.

* Frequency cross-talk—The Genome Analyzer uses two lasers and four filters to detect four dyes attached to the four types of nucleotide, respectively. The frequency emission of these four dyes overlaps so that the four images are not independent. The frequency cross-talk is deconvolved using a frequency cross-talk matrix.

* Phasing/Prephasing—Depending on the efficiency of the fluidics and the sequencing reactions, a small number of molecules in each cluster may run ahead (prephasing) or fall behind (phasing) of the current incorporation cycle. This effect is mitigated by applying corrections during the base calling step.

* All of these corrections are based on an assumption of '''equal base frequency.''' For this reason, Illumina recommends the inclusion of the PhiX control sample in all runs (7+1). Work is currently under way at the [Broad Institute] to create defined spiked in DNA templates with equal base frequency that can act as control reads, however, this has not yet been incorporated into the pipeline.