Ribo-seq analysis workflow

Question

[mansiparihar13]

I am working on Ribo-seq analysis workflow and the sample that I am using is a paired end sample, so when I do the pre-processing steps like fastqc, trimming then postqc , the results I am getting are not good quality maybe because the sample is paired end and paired end samples get overlapped while processing, then I tried with only one read and the results I got were good quality results. So, for Ribo-seq profiling can we use single reads or the paired end is required? I reviewed some literature also on this, and in many papers single end processing is being carried out

Answer 1

[Kevin Blighe]

Yes, single-end reads are sufficient and widely used for Ribo-seq profiling, as ribosome footprints are typically short (~26–34 nt), making paired-end sequencing unnecessary for most analyses. Many pipelines (e.g., nf-core/riboseq) support both, but single-end avoids overlap issues in short fragments and is routine in literature. Stick with your single-end results—they're valid. If needed, tools like Trimmomatic with PE mode and overlap handling can fix paired-end QC.

Kind regards, Kevin