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Dear Audrey,

I am interested in analyzing RPF data, and I have been checking your GWIPS-viz database. It's a really nice job putting all these data together for the scientific community!

I have a couple of questions regarding your database, it would be great if you could help me with them!

1. In some cell lines (e.g HeLa from Guo, PC3 from Hsieh), you have three different tracks: mRNA, ribosome coverage and ribosome profiles. However, which is the difference between ribosome coverage and ribosome profiles?
2. Following the same question, how come that in other cell lines (e.g. HeLa from Stadler and Fire) there is not both ribosome coverage and ribosome profiles (just ribosome coverage)?
Dear Eva,

Thanks very much for your interest in GWIPS-viz. My answers to your questions are in red below:

1. In some cell lines (e.g HeLa from Guo, PC3 from Hsieh), you have three different tracks: mRNA, ribosome coverage and ribosome profiles. However, which is the difference between ribosome coverage and ribosome profiles?
Typically a ribosome footprint is ~30 nucleotides. A ribosome coverage plot represents the nucleotide sequence that the ribosome footprint covers from the 5' end to the 3'end (~30nts). So the coverage plots in GWIPS-viz show the number of read counts that covers a given base in the reference sequence.
For the ribosome profiles, only the location where the 5' end of the footprint aligns to is counted (and not the entire footprint). For the ribosome profiles of elongating ribosomes, we use a 15nt offset from the 5' end of the read so that the footprint alignments represent the A-site of the ribosome. For ribosome profiles of initiating ribosomes we us a 12nt offset for the P-site.

2. Following the same question, how come that in other cell lines (e.g. HeLa from Stadler and Fire) there is not both ribosome coverage and ribosome profiles (just ribosome coverage)?
We plan to add ribosome profiles for all the datasets for which we provide coverage plots. In fact, we hope to provide them very soon. And indeed we will also incorporate other publicly available ribosome profiling datasets.
Audrey and Eva,

Thanks for this post, this is very helpful- with regards to the A- and P-sites- what is the decision point to use either the A- or P-site for peak calling? Some papers just use only the A-site or just the P-site... I appreciate your insights in advance,

(14-Aug-2013 10:08 AM)audrey Wrote: [ -> ]Dear Eva,

Thanks very much for your interest in GWIPS-viz. My answers to your questions are in red below:

1. In some cell lines (e.g HeLa from Guo, PC3 from Hsieh), you have three different tracks: mRNA, ribosome coverage and ribosome profiles. However, which is the difference between ribosome coverage and ribosome profiles?
Typically a ribosome footprint is ~30 nucleotides. A ribosome coverage plot represents the nucleotide sequence that the ribosome footprint covers from the 5' end to the 3'end (~30nts). So the coverage plots in GWIPS-viz show the number of read counts that covers a given base in the reference sequence.
For the ribosome profiles, only the location where the 5' end of the footprint aligns to is counted (and not the entire footprint). For the ribosome profiles of elongating ribosomes, we use a 15nt offset from the 5' end of the read so that the footprint alignments represent the A-site of the ribosome. For ribosome profiles of initiating ribosomes we us a 12nt offset for the P-site.

2. Following the same question, how come that in other cell lines (e.g. HeLa from Stadler and Fire) there is not both ribosome coverage and ribosome profiles (just ribosome coverage)?
We plan to add ribosome profiles for all the datasets for which we provide coverage plots. In fact, we hope to provide them very soon. And indeed we will also incorporate other publicly available ribosome profiling datasets.
It does not really matter since 'a-site'='p-site'+3
IMO showing locations of a-sites is more natural because these are the codons that are being decoded by the ribosomes that leave footprints.
However, the most popular method for determining offset is looking at the distance between the high peak at the 5' end of the metagene profile relative to the location of the start codon. This distance is assumed to be the distance between the P-site codon and 5' end of the footprint, because the initiator tRNA is incorporated into the p-site, not the a-site. So for some people it might be more natural to show p-site locations for all the ribosomes.
I don't think it really matters as long as you don't mix the two.


(04-May-2016 08:19 PM)chrishlee23 Wrote: [ -> ]Audrey and Eva,

Thanks for this post, this is very helpful- with regards to the A- and P-sites- what is the decision point to use either the A- or P-site for peak calling? Some papers just use only the A-site or just the P-site... I appreciate your insights in advance,

(14-Aug-2013 10:08 AM)audrey Wrote: [ -> ]Dear Eva,

Thanks very much for your interest in GWIPS-viz. My answers to your questions are in red below:

1. In some cell lines (e.g HeLa from Guo, PC3 from Hsieh), you have three different tracks: mRNA, ribosome coverage and ribosome profiles. However, which is the difference between ribosome coverage and ribosome profiles?
Typically a ribosome footprint is ~30 nucleotides. A ribosome coverage plot represents the nucleotide sequence that the ribosome footprint covers from the 5' end to the 3'end (~30nts). So the coverage plots in GWIPS-viz show the number of read counts that covers a given base in the reference sequence.
For the ribosome profiles, only the location where the 5' end of the footprint aligns to is counted (and not the entire footprint). For the ribosome profiles of elongating ribosomes, we use a 15nt offset from the 5' end of the read so that the footprint alignments represent the A-site of the ribosome. For ribosome profiles of initiating ribosomes we us a 12nt offset for the P-site.

2. Following the same question, how come that in other cell lines (e.g. HeLa from Stadler and Fire) there is not both ribosome coverage and ribosome profiles (just ribosome coverage)?
We plan to add ribosome profiles for all the datasets for which we provide coverage plots. In fact, we hope to provide them very soon. And indeed we will also incorporate other publicly available ribosome profiling datasets.
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