Why Are There Differences in RNA-seq Results?

Question

[ZZOYEA]

Hello, I am analyzing total RNA-seq results and checking them in IGV.

I have noticed that the results from our experimental samples are different from the public data downloaded from GEO. The top 6 tracks are from our experimental samples, and all the tracks below the reference are from the public data.

What I am curious about is why there are small read counts between the exons in our data, but in the public data, which used the same cell line and the same drug treatment, there are none and the results are clean. At which stage do these differences arise? Should we consider the small read counts in our samples as noise?

[BioinfGuru]

Assuming all display parameters are equal.

It also looks like all peaks across the whole length of the image are higher in your dataset. Have you compared overall sequencing depth between the local/public data sets?

[ZZOYEA]

No, not yet.. I'll check that out. Thank you!!

Answer 1

[jared.andrews07]

Intronic reads as seen here are pretty normal in total RNA-seq, as you're capturing more nascent transcripts than you would after polyA selection. Sometimes, these reads can be a pretty significant fraction of total reads, especially in certain tissues (e.g. brain) and developmental context (tend to be more in fetal than adult).

Is the data in GEO also total RNA-seq or is it polyA selected? It looks like sequencing depth may also just be lower. Generally, we do not throw out intronic reads. This wouldn't concern me too much, my guess is the differences stem from library prep method or sequencing depth.

[ZZOYEA]

As I know the GEO data is total RNA-seq, but I need to look into the method section a bit more. Thank you!!