Notes on the MLOGD 'Six-frame' plot:

This is a plot of the MLOGD statistic calculated in a sliding window along the alignment in each of the six possible read-frames. In each window, the likelihood ratio that 'Both the window and the input Known CDS(s) are coding' versus 'Only the input Known CDS(s) are coding' is calculated and summed over the phylogenetic tree (as described here). The sixteen panels show the following information:

1. This panel shows the positions of alignment gaps in each of the input sequences (labelled at right).
2. This panel shows the positions of stop codons in each of the six possible read-frames in each of the input sequences (labelled at right).
3. This panel shows the likelihood ratio score in each window in the +0 frame (relative to reference sequence nucleotide 1), summed over the input sequence pairs. The width of the window is indicated by the horizontal grey line. The window width is determined on the reference sequence, so if the reference sequence contains alignment gaps within the window, then the window will appear larger in alignment coordinates. The dashed line is at zero.
4. This panel shows the positions of stop codons in the +0 frame in all the input sequences (same order as in panel 1).
5. As panel 3, +1 frame.
6. As panel 4, +1 frame.
7. As panel 3, +2 frame.
8. As panel 4, +2 frame.
9. As panel 3, -0 frame.
10. As panel 4, -0 frame.
11. As panel 3, -1 frame.
12. As panel 4, -1 frame.
13. As panel 3, -2 frame.
14. As panel 4, -2 frame.
15. Input Known, or null model, CDS(s).
16. This panel shows the phylogenetic sum of sequence divergences (mean number of mutations per nucleotide) for the sequence pairs that contribute to the likelihood ratio sum at each position in the alignment. In any particular column, some sequences may be omitted from the likelihood ratio calculations due to gaps or stop to non-stop transitions. Statistics in regions with lower summed divergence (i.e. partially gapped regions) have a lower signal-to-noise ratio.

Notes:

• In general, you wouldn't expect to have in-frame stop codons within any of the annotated CDSs, since the annotated CDSs should in general be conserved across the alignment. A few stops near the ends of CDSs are not unusual and indicate that the CDS terminates early in some sequences. However, if there are many in-frame stop codons within the annotated CDSs, then this may indicate a CDS annotation or alignment problem.
• Note that the scores in the plots have been summed over the input pairs file or phylogenetic tree. If a CDS is present in some, but not all, of the input sequences, then the pattern of mutations consistent with coding in some of the sequences will be diluted by the pattern of mutations consistent with non-coding in the other sequences.
• Note that alignment problems may cause a non-reference sequence codon to be aligned out-of-frame to a reference sequence codon. This may occassionally result in an out-of-frame non-reference sequence stop codon being incorrectly annotated on the plot. This can be avoided by keeping gaps in groups of three within CDSs (see also this note). Therefore, if you have an isolated stop codon in what otherwise appears to be a long conserved ORF, you should check that it is not the result of a local alignment problem.
• Note also that in places where the reference sequence contains alignment gaps, there is no frame information for the non-reference sequences. As far as calculation of statistics is concerned, all such regions are omitted. However for the stop and start codon annotation, any non-reference sequence stops or starts within reference sequence gaps will be missed.
• For panels 3, 5, 7, 9, 11 and 13, the score for a particular window may be omitted from the plot if it is partially or total gapped in some or all of the sequence pairs. Scores are omitted if the sum, S, over sequence pairs of (number of nt used in window) x (pairwise sequence divergence) is less than some threshold value (details). Scores from partially gapped regions which are not omitted, are scaled by S_max / S (details).