frames

[utahmike]

Hi Ribogalaxy team, I am pushing through the riboseqR analyses for the first time. I am slightly confused about the frames aspect in metagene analysis. First, I understand that the field "Length of ribosome footprint reads to be used in filtering (lengths):" refers to what size sequence reads one is interested in. In the next field, "Frames of the ribosome footprint reads to be used in filtering (frames):" i assume this is referring to the frame in which the 5' end of the sequence read maps. If I want only 1 size (30 nts) and only those with 5' end mapping to 1 reading frame (0) then I would enter:

Length of ribosome footprint reads to be used in filtering (lengths): 30
Frames of the ribosome footprint reads to be used in filtering (frames): 0

If I want all 3 frames considered for one size sequence read then I would enter:
Length of ribosome footprint reads to be used in filtering (lengths): 30,30,30
Frames of the ribosome footprint reads to be used in filtering (frames): 0,1,2

Is this correct?

The output of this then becomes the input for differential translation analysis. If I only enter 30 and 0 in step 3, the metagene analysis, will I be able to specify other sizes and other reading frames in the differential translation analysis?

Thanks in advance,
Mike

[audrey]

Hi Mike,

For the metagene plot, the value(s) entered for the 'Frames of the ribosome footprint reads to be used in filtering' are usually taken from the triplet periodicity results where the likely frame for individual read lengths (frame.ML) are provided in the 'Results of reading frame analysis' section (at the top of the page).

So if you want to generate a metagene plot for one read length size (e.g 30 nts) and the corresponding likely frame (frame.ML) is 0, then yes what you suggested below is correct:

Length of ribosome footprint reads to be used in filtering (lengths): 30
Frames of the ribosome footprint reads to be used in filtering (frames): 0

The reads of length 30 nts that are not mapping to the "dominant" frame will still be plotted in the metagene plot.

As the differential analysis tool input is the metagene output, only the reads considered in the metagene analysis will be considered for the differential analysis. But you could run a separate metagene analysis to include all appropriate reads ( e.g 28, 29, 30, 31, 32 and the corresponding frame.ML 2, 2, 2, 2, 1)
And then run the differential analysis on this metagene file output.

Could you try this and let us know if it works for you?

Audrey