Hi,

I'm new to Ribo-Seq and trying to understand how the analyze the data. I have difficulties understanding what the offset means. I know it is defined as the distance between the start of the read at the 5'-end and the P-site, which is inside the ribosome. 

But how is this offset calculated? why do I have sometimes different offset values for different read lengths, but sometime not and why do i use different offsets for the same read length in different data sets?
Is there a tutorial or a workflow explaining this approach?

thanks
Hi Assa, 

Apologies for making you wait so long for a response. I hope this answer can still be of some use to you. 

You are correct with your definition of the Offset for the most part but I believe we can be a small bit more accurate. I think of the offset as the distance from one end of the Ribo-Seq read to the position of the decoding centre of the ribosome on the fragment. Depending on the dataset it may be optimal to calculate offset from the 3'  end and the offset value will vary as a result. For elongating ribosomes, such as those isolated when cyclohexamide is used to halt translation we may prefer to calculate offset to the A-site. 

There are a number of approaches that have been used to calculate the offset. Historically, set offset lengths were employed for a whole sample depending on the samples treatment. Elongating ribosomes got a set offset of 15 and initiating got a set offset of 12. This was in order to target the P-site for initiating ribosomes and the a-site for elongating 

However, this approach is not optimal as the true distance to the decoding centre will differ depending on the length of the fragment. I think Figure S2 in the [ORFik publication][1] is a nice visualisation of this. And a typical calculation of 5' and 3' offset is described in the [Shoelaces paper][2]. 


  [1]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8214792/
  [2]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6052522/