Hello all,
I extracted RNA from my samples and performed RIP-seq. After annotating the genomic regions using R, I obtained promoters, exons, introns, and UTRs. Given that my samples consist of RNA, and RNA molecules themselves do not have promoters (which are specific to DNA sequences), I am puzzled as to why promoters appeared in the results. What could be the cause of this, and how should I proceed?
Thank you
There are 3 potential explanations.
1. You have some DNA contamination in your pulldown.
2. Annotations are not correct.
3. Many promoters are transcribed bi-directionally, the main transcript and promRNAs that are usually short, unstable transcript expressed in the antisense direction.
If your library prep preserve the strand information than if option 3 is the case you should see that your promoter mapped regions are expressed in the opposite direction to the adjacent annotated transcript.
It appears that the package you're using is meant for DNA analysis and is detecting DNA-associated patterns like promoters, exons, and introns. RNA-seq data requires specialized tools to accurately annotate RNA transcripts. Here's a full, detailed guide on how to proceed:
Switch to RNA-specific Annotation Tools
To properly annotate your RNA-seq data and avoid the misannotation of promoters and other DNA-specific features, you should use tools designed for RNA analysis. Here’s a guide on how to proceed:
1. Align RNA Reads
Tool: HISAT2
- Purpose: Align RNA-seq reads to the reference genome.
2. Assemble Transcripts
Tools: StringTie or Cufflinks
- Purpose: Assemble aligned reads into potential RNA transcripts.
3. Merge Assemblies
Tool: StringTie
- Purpose: Merge transcript assemblies from multiple samples to create a unified transcript annotation.
4. Quantify Transcripts
Tools: StringTie or Cufflinks
- Purpose: Quantify the expression levels of assembled transcripts.
5. Annotate Transcripts
Tool: GffCompare or other RNA-specific annotation tools
- Purpose: Compare assembled transcripts to a reference annotation to identify known and novel transcripts.