I am interested in doing a Ribo-Seq analysis and although I have searched a lot (without success) about how many reads are recommended for mRNA and RPF in order to perform a TE analysis... I have ended with even more doubts.
Some papers say that they sequence more million reads for mRNA than RPF, others more millions in RPF than mRNA... and others (at least 1) that they sequenced the same amount of reads for both.
I know that it is required to have at least 20 million of mapped reads in RPF... and RPF needs to have more sequencing depth due to contamination and that they do not align as much as mRNA. But... if I want to calculate the translation efficiency (TE) = RPF/mRNA, if I have more reads aligned to RPF than mRNA or viceversa, wouldn't I over/under estimate some genes? Wouldn't it make more sense to have the same amount of aligned reads for both (mRNA and RPF)?
These are some papers that they have more reads in mRNA than RPF:
- https://doi.org/10.1093/nar/gkaa1292
- https://doi.org/10.1093/nar/gkz633
- https://doi.org/10.1007/s13238-018-0535-6
- https://doi.org/10.1182/bloodadvances.2020001717
These are some papers that they have more reads in RPF than mRNA:
- https://doi.org/10.3389/fmicb.2021.781454
- https://doi.org/10.1128/msystems.00126-18
- https://doi.org/10.1093/nar/gkz471
- https://doi.org/10.1158/0008-5472.CAN-18-0631
This is the only paper that I found with similar amount of reads for both:
What do you think? Any opinion/feedback will be well received.
Thanks very much in advance
