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I am working on RNA seq analysis and I would like to know the following things:

Current Methods:

  • downloaded genome fasta file for non-coding rna from ensembl and got the gtf file for hg38
  • performed hist2 and got 17% alignment for my sample against ncrna genome
  • sorted the file and then ran feature counts with the hg38 gtf file but got zero counts

Question:

  • what could be the issue that is causing zero counts?

My bam file after sorting has the SRR ID at first whereas the GTF file has chromosome no: . Could this be the issue?

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    $\begingroup$ Please clarify your specific problem or provide additional details to highlight exactly what you need. As it's currently written, it's hard to tell exactly what you're asking. $\endgroup$
    – Community Bot
    Commented Nov 25, 2023 at 7:37
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    $\begingroup$ Did you spot-check where the alignments are? $\endgroup$
    – Devon Ryan
    Commented Nov 28, 2023 at 14:53
  • $\begingroup$ nope. i guess there was an issues with the refernce genomecompatibility. $\endgroup$ Commented Dec 1, 2023 at 3:04

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Several issues:

  1. There is no non-coding RNA genome. There are non-coding genes but the genome fasta files from Ensembl are always the entire genome and this is what you should align against. Probably you downloaded a transcriptome fata file.

  2. If you aligned against a transcriptome fasta file (like ncRNAs) then it makes no sense to use a GTF file since GTF files have genome coordinates but you mapped against a selection of a transcriptome fasta, hence you have (specific) transcriptome coordinates. GTF and these coordinates are not compatible.

Solution: Align your data against the entire genome (hg38), then use the GTF to get counts for all annotated genes, then do subsetting to specific genes if that is what you need.

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  • $\begingroup$ Thank you so much. I guess i have been using transcriptome fasta file. will change that . $\endgroup$ Commented Dec 1, 2023 at 3:03

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