Note on read-frame combinations that can lead to false positive signals:

The following read-frame combinations can sometimes result in false positive signals (read-frame notation):

• A query ORF overlapping a known CDS, with the query ORF in the -2 frame relative to the +0 frame known CDS, is liable to produce a positive signal even when the query ORF is non-coding. This is because, in the -2 frame, N3 (the 3rd nucleotide position in codons) in the query ORF aligns with N3 in the known CDS. The pattern of mutations for two CDSs overlapping in these frames is 'highly variable N3 and constrained N1 and N2' (the 1st and 2nd nucleotide position in codons). This is similar to the pattern of mutations for a single non-overlapping CDS.
  
  In the six-frame plots, the read-frame combinations to be wary of are:
  
  

  Known CDS (coding)	Query CDS (false +ve)
  +0	-2
  +1	-0/-3
  +2	-1
  -0/-3	+1
  -1	+2
  -2	+0

  
  The presense or absense of stop codons can be a useful additional clue as to whether such a region of positive signal is or isn't coding.
  
  
• When there are two overlapping known CDSs, in the overlap region only the first CDS in the input 'Known CDSs' list will be incorporated into the null model. In the six-frame plots, the other CDS should show up as a positive signal. In addition, there are certain read-frame combinations of the two known CDSs that may lead to a false positive signal in a third frame. These are as follows (for Known CDS 1 in the forward read-direction):
  
  

  Known CDS 1 (coding)	Known CDS 2 (coding)	Query CDS (false +ve)
  +0	-0/-3	+1
  +0	+1	-0/-3
  +0	+2	-1
  +0	-1	+2
  +1	-1	+2
  +1	+2	-1
  +1	+0	-2
  +1	-2	+0
  +2	-2	+0
  +2	+0	-2
  +2	+1	-0/-3
  +2	-0/-3	+1

  
  The presense or absense of stop codons can be a useful additional clue as to whether such a region of positive signal is coding. Aside from that, while these particular read-frame combinations may give a false positive signal when the Query CDS is tested against Known CDS 1, they are all expected to give a negative signal when the Query CDS is tested against Known CDS 2, and thus may be selected out in this way.