1. For this pipeline, should we be using Trim Galore --length and --max_length to have cutoffs for Ribosome profiling? And if so, would 17-34nt be correct (ref Ingolia 2017 Methods paper https://pubmed.ncbi.nlm.nih.gov/28579404/)? 2. I had created a max length filter of 30, as one of my samples had few longer than 30nt and I wanted to be consistent across all samples. When I do this, the pipeline breaks at the Salmon quant step:

[2025-06-13 18:28:16.677] [jointLog] [warning] salmon was only able to assign 0 fragments to transcripts in the index, but the minimum number of required assigned fragments (--minAssignedFrags) was 10. This could be indicative of a mismatch between the reference and sample, or a very bad sample.  You can change the --minAssignedFrags parameter to force salmon to quantify with fewer assigned fragments (must have at least 1).

I believe this is due to kmer size being 31 by default? This got me thinking that regardless of whether I have filters or not, for salmon indexing and quantification, should I change it to something smaller to best reflect our smaller fragments?

Hi, can I ask how did you resolve this issue? I’ve tried preparing a new index with kmer being 23, but the problem persisted. Looking forward to hearing about your solution