Hello,
I work with a non model organism (fish) and I am approaching miRNA analyses for the first time. Most of the papers I have read remove tags that align to exons, introns and repeat sequences of the reference genome, and use the unaligned tags to identify conserved and novel miRNAs. Could anyone please explain me why they remove these tags? I have read that a good proportion of miRNAs originates from intronic and exonic regions of other genes, so why should we get rid of them?
Removing tags aligning to exons and introns is a common preprocessing step in miRNA (microRNA) analysis for several reasons:
Focus on miRNAs: The primary goal of miRNA analysis is to identify and quantify miRNAs, which are short non-coding RNAs involved in post-transcriptional gene regulation. Exons and introns are parts of protein-coding genes, and tags aligning to these regions do not represent miRNAs. Removing them helps to focus the analysis specifically on miRNAs of interest.
Reducing noise: Sequencing data can contain various types of noise and artifacts. Tags aligning to exons and introns may arise due to background noise, experimental artifacts, or RNA degradation products. Removing these non-miRNA tags reduces noise and increases the accuracy of miRNA quantification.
Avoiding misinterpretation: Including tags from exons and introns in miRNA analysis can lead to misinterpretation of the results. For example, if you count tags from these regions as miRNAs, it can lead to overestimation of miRNA expression levels and false positives.
Streamlining downstream analysis: Many downstream miRNA analysis tools and pipelines are designed to work with data that only contains miRNA-specific reads. Removing tags aligning to exons and introns makes the data more compatible with these tools and simplifies the analysis process.
To achieve this, researchers typically use alignment tools like Bowtie, STAR, or HISAT2 to map sequencing reads to a reference genome, and then they filter out reads that align to exonic and intronic regions before proceeding with miRNA-specific analysis. This preprocessing step helps ensure that the subsequent analysis is focused on the biologically relevant miRNAs and provides more accurate insights into their expression and function.
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