High intronic mappings in the RNASeq
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2.2 years ago

Hi, I have some samples from a frozen tissue bank, extract the RNA and sent them to RNASeq. The result mappings are weird to me, with 30~40% mapping rate by Salmon. I tried hisat2, the mapping rate goes to >80%. samtools sort the sam files to bam, and them qualimap2 gives me the QC results:

Exonic: | 31,212,828 / 41.39%

Intronic: | 39,191,136 / 51.97%

Intergenic: | 5,008,406 / 6.64%

Intronic/intergenic overlapping exon: | 6,243,753 / 8.28%

There is not too much DNA contamination, but a large portion of intronic mappings. Is there anyone who knows how to interpret this result? What can I do with this RNASeq results?

Any advice is welcome.

Best regards, Raymond

RNA-Seq • 941 views
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Depends on the organism and how well the genome is annotated, as well as whether you used polyA or ribo depletion libraries. So providing more details would be helpful

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I used human hg38 Ensembl release 84. ribo depletion is used

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This paper suggests ployA method is much better method to use https://www.nature.com/articles/s41598-018-23226-4

Also I'd like to point out that Salmon and HTseq seem to agree. About ~40% of your reads are exonic. How's the coverage across the exonic regions? If they're good then you can continue as normal IMO, that's assuming you performed deep sequencing.

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That does seem a bit weird - are they nuclear?

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