Bandcalling

Using the lane-tracking information generated in the previous stage, the band calling algorithm involves the following steps:

  • Trace extraction and preparation.
  • Locating the well.
  • Then for each lane.
    • Locate peaks in the trace.
    • Filter peaks to extract bands.

Trace preparation

It is desirable to subtract the background intensity from the trace prior to band-calling. This simplifies the modeling of bands and improves the visualization for the user.

Finding the well

A specific algorithm was introduced to locate the feature corresponding to the point on the gel where the DNA was loaded. This feature runs across the gel and, although superficially like a band, it can be sufficiently different to justify a specific algorithm for its detection. Prior information about the approximate location of the well is combined with an algorithm that looks for a drop in intensity to either side of the well.

Locate peaks in the trace

The trace is examined and local peaks are identified. Where the trace has a plateau, an interval where the trace is equal to the local maximum, the peak is taken to be the central point of the plateau.

Filter peaks to extract bands

A filter which models the expected band height is then applied to remove the effects of local noise. The filter is exponentially decaying and only those peaks which are greater than the filter are retained. The filter parameters are determined from the band height model. To exclude the additional band called in the 'noisy' tail of the lane the last 5% of the bands are rejected.

* quick link - http://q.sanger.ac.uk/70tnm0zf