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From a RNA seq experiment we look for the counts of transcripts of tRNA but we are also interested of other types of RNA we can find. Because of the protocol used for the library prep we can't analyse miRNA but we can analyse ncRNA. I never analyzed transcripts for ncRNA and I have some questions:

  • The name of the transcripts are like ENST00000611544.1, ENST00000387069.1, since they are non coding, are they linked to a gene? Should I rename the name of each ncRNA with a gene name or working with these long names is ok?
  • When analyzing transcripts of RNA I did enrichment analysis to see what pathways are more active. But when analyzing transcripts for ncRNA is possible to do something similar to enrichment analysis? How can analyse the functions of the ncRNA that we get from the study?

Thank you very much in advance

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  • $\begingroup$ Hi @Mee it might be that a RNAseq expert understands your concerns precisely, however could you provide a bit more background for everyone else please? For example, what package are you performing the analysis with, any description of the samples? $\endgroup$
    – M__
    Aug 21 '20 at 12:26
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ncRNAs are by itself not special. Sure they might be more unstable and less abundant than other transcripts but for the analysis it is more or less the same. You should take your transcript level counts and aggregate them to the gene level. The tximport package comes to mind. Every transcript (given it is annotated) is linked to a gene. ENST00000611544 is connected to the U2 gene but these snRNAs are also short and I doubt you see them in normal RNA-seq.

In the end you can process data as any RNA-seq. Quantify your data against a transcriptome, e.g. with salmon, then aggregate transcript level abundance estimates (so basically counts) to the gene level and see whether your genes of interest have counts you can work with or whether you mostly have zeros. A transcriptome can be obtaine e.g. from GENCODE. The available annotation files will tell you which genes these transcripts originate from. Check for the GTF files.

Towards enrichment analysis, well it all comes down to the annotations you have. If the ncRNAs are annotated in any pathway then you can perform standard analysis with tools such as goseq or gprofiler2. If they are not annotated, well then there is no information available.

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