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I am studying about RNA seq. I have found that in 3'prime rna seq (quantseq, tagseq) we should use a reference genome with good annotation (plus known UTRs). My question is why? Why could not we use reference transcriptome? Thank you!

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    $\begingroup$ You asked a similar question before bioinformatics.stackexchange.com/questions/14093/… and were told to use the genome. If you so badly want to align to the transcriptome then do that but stop asking us for advice. Asking something when you are already convinced of an answer is a waste of time. Repeating the question when you don't like the answer is just plain rude. $\endgroup$ Aug 8, 2020 at 14:28

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It would be great if you can elaborate on where you "found" this information.

In the transcriptome annotation you have a defined number of transcript per genes, but there might be others that are present in your sample and not in the annotation. For example you have treated the cells with some extreme condition that changes the 3' processing machinery.

With 3' quantseq, you are left with the read fragment that is at the end of your transcript. If the ends of these transcripts are not part of the sequences in the annotated transcriptome, these will be unaligned.

These instances might be rare if your transcriptome annotation is good, but let's say if you find that a gene of interest which should be expressed is not, and you suspect it might be due to a different isoform used, you can always check it with the genome alignment (see where reads might be falling in the gene). With a transcriptome alignment, this will not be possible.

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I'm not sure that's true:

https://www.nature.com/articles/s41598-019-55434-x

"QuantSeq. 3′ Sequencing combined with Salmon provides a fast, reliable approach for high throughput RNA expression analysis"

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  • $\begingroup$ Yes I have seen this paper..thank you..but on another reliable source I have found the piece of information that I posted.. $\endgroup$
    – marilu
    Aug 7, 2020 at 22:22

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