<p>As @DevonRyan mentioned, it's very likely that those samples were degraded, which is good justification for excluding them from subsequent analysis.</p>

CC BY-SA 4.0
2019-01-09T15:06:27.920Z
2019-01-09T15:06:27.920Z
Daniel Standage
<p>I would expect at least 30% of reads from a total-cell, ribo-depleted RNA-seq to be exonic. Less suggests something when wrong. </p>

<p>As well as degradation, another explanation would be contamination with genomic DNA. </p>

<p>RNA obtained from the nuclear or cytoplasmic fraction of the cell might have a exonic content lower than 30%, while poly-A selected RNA would be expected to have higher, more like 66%. </p>

CC BY-SA 4.0
2019-01-10T12:26:16.717Z
2019-01-10T12:26:16.717Z
Ian Sudbery
<p>After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason?</p>

<pre><code>Samples   Exonic            Intronic    Intergenic
Sample1 545479 (12.8%)  2512309 (58.8%) 1217201 (28.5%)
Sample2 372836 (8.3%)   2556934 (56.8%) 1573032 (34%)
Sample3 529618 (7.3%)   3934615 (54.5%) 2750720 (38.1%)
</code></pre>

<p>I also see that 35-40k genes among 58k genes having "zero" read counts. Is this due to contamination? What could be the reason? What does reads mapping to exonic, intronic, intergenic tell?</p>

<p>Update: I used hisat2 for alignment. I used human genome "grch38_snp_tran" from hisat2 website. Libraries were generated using ribosome depletion kit.</p>

CC BY-SA 4.0
2019-01-09T15:15:10.467Z
There is the potential that these samples had low quality RNA or genomic DNA contamination (depending on the sample type and RNA isolation method)
GWW
CC BY-SA 4.0
2019-11-24T12:06:28.883Z
What software did you get this output from?
Aliakbar Hasankhani
CC BY-SA 4.0
2018-05-07T19:40:21.413Z
How did the overall alignment rate of those samples compare to the others that had more reasonable intronic alignment rates?
Devon Ryan
CC BY-SA 4.0
2018-05-07T20:12:41.473Z
I see that all the 37 samples have overall alignment rate between 90-96% and for those 3 samples it is 69%, 79%, 70% overall alignment rate.
stack_learner
CC BY-SA 4.0
2018-05-07T20:16:21.107Z
Sounds like you have degraded samples
Devon Ryan
CC BY-SA 4.0
2018-05-07T20:34:16.603Z
U mean those 3 samples are degraded? What I should do now? Should I remove them for further analysis?
stack_learner
CC BY-SA 4.0
2018-05-07T20:35:28.097Z
It’s a likely cause, but regardless you should exclude them.
Devon Ryan
CC BY-SA 4.0
2018-05-07T20:55:44.087Z
Sure. the high percentage of mapping to intronic region is due to degradation?
stack_learner
CC BY-SA 4.0
2018-05-08T06:58:54.547Z
Could you add the quality metrics of these files and other of the same run to the question?
llrs
<rna-seq><phylogenetics><rna><reads>
Reads mapped to exonic, intronic and intergenic regions
# Reads mapped to exonic, intronic and intergenic regions

(Asked by: username_0 on 2018-05-07)

After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason?
Samples   Exonic            Intronic    Intergenic
Sample1 545479 (12.8%)  2512309 (58.8%) 1217201 (28.5%)
Sample2 372836 (8.3%)   2556934 (56.8%) 1573032 (34%)
Sample3 529618 (7.3%)   3934615 (54.5%) 2750720 (38.1%)

I also see that 35-40k genes among 58k genes having "zero" read counts. Is this due to contamination? What could be the reason? What does reads mapping to exonic, intronic, intergenic tell?
Update: I used hisat2 for alignment. I used human genome "grch38_snp_tran" from hisat2 website. Libraries were generated using ribosome depletion kit.

**Tags:** rna-seq, phylogenetics, rna, reads
> **Comments:**
> **username_3**: How did the overall alignment rate of those samples compare to the others that had more reasonable intronic alignment rates?  
> **username_0**: I see that all the 37 samples have overall alignment rate between 90-96% and for those 3 samples it is 69%, 79%, 70% overall alignment rate.  
> **username_3**: Sounds like you have degraded samples  
> **username_0**: U mean those 3 samples are degraded? What I should do now? Should I remove them for further analysis?  
> **username_3**: It’s a likely cause, but regardless you should exclude them.  
> **username_0**: Sure. the high percentage of mapping to intronic region is due to degradation?  
> **username_4**: Could you add the quality metrics of these files and other of the same run to the question?  
> **username_1**: There is the potential that these samples had low quality RNA or genomic DNA contamination (depending on the sample type and RNA isolation method)  
> **username_2**: What software did you get this output from?  

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## 2 answers

### Answer by username_5 on 2019-01-09. Score: 3

As @DevonRyan mentioned, it's very likely that those samples were degraded, which is good justification for excluding them from subsequent analysis.


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### Answer by username_6 on 2019-01-10. Score: 2

I would expect at least 30% of reads from a total-cell, ribo-depleted RNA-seq to be exonic. Less suggests something when wrong.

As well as degradation, another explanation would be contamination with genomic DNA.

RNA obtained from the nuclear or cytoplasmic fraction of the cell might have a exonic content lower than 30%, while poly-A selected RNA would be expected to have higher, more like 66%.


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