<p>At least with Ensembl annotations, you can <code>grep rRNA foo.gtf &gt; rRNA.gtf</code> to get the annotated rRNAs. Note however, that the <a href="https://www.ncbi.nlm.nih.gov/gene/100861531" rel="nofollow noreferrer">45S cassette</a> isn't in the mouse reference genome, so you'll never have alignments to 18S, 5.8S, or 28S rRNA. I always get the feeling that the Collect*Metrics programs are made with human sequences in mind, since the cassette is included in the human reference genome.</p>

<p>Anyway, while you can use the annotation and reference sequence to make picard happy, make sure to append the Rn45S sequence to your genome if you want to actually measure rRNA levels. In my experience this is only useful for rRNA-depleted samples, since the rates can vary wildly there.</p>

<p><strong>Edit</strong>: To make a file compatible with picard's "interval list", one can do the following with the GTF file:</p>

<pre><code>grep rRNA foo.gtf | awk 'BEGIN{OFS="\t"}{print $1, $4-1, $5}' &gt; foo.bed
</code></pre>

<p>According to the <a href="https://gatkforums.broadinstitute.org/gatk/discussion/1319/collected-faqs-about-interval-lists" rel="nofollow noreferrer">picard documentation</a> a BED file can be used as input and the above <code>awk</code> code extracts the appropriate columns and switches to 0-based coordinates used in the BED format.</p>

CC BY-SA 3.0
2017-07-19T21:14:01.107Z
Interesting, can I directly use the output of `grep rRNA foo.gtf > rRNA.gtf` as input file? or is there any further modification necessary? I did not understand when you say `make picard happy` I am using mm9 reference and yes the sequencing done was using ribo depleted technique (could you please point towards some literature or steps)
novicebioinforesearcher
CC BY-SA 3.0
2017-07-19T21:20:50.707Z
@novicebioinforesearcher I've updated my reply with the conversion to BED, which allegedly should work. "make picard happy" is another way of saying "give picard a file it can use as input". The results would still not be terribly useful without adding in the Rn45S sequence.
Devon Ryan
CC BY-SA 3.0
2017-07-19T21:28:15.753Z
I never came across Rn45S concept (thanks for pointing it out) and have not seen any literature in the tools that use such list what would be your thoughts as to why they would not be speak about it?
novicebioinforesearcher
CC BY-SA 3.0
2017-07-19T21:31:08.847Z
@novicebioinforesearcher Many reference genomes (in particular, human and fly) contain it already, so there'd be no need to use Rn45S there. I don't know why it was excluded from the mouse genome, it's one of the things that's always bothered me.
Devon Ryan
CC BY-SA 3.0
2017-07-19T21:08:28.883Z
2017-07-19T21:20:24.753Z
2017-07-19T21:20:24.753Z
Devon Ryan
<p>Could some one please help me with understanding how to generate the ribosomal interval list that is required to when using picard metrics this step</p>

<pre><code>java -jar picard.jar CollectRnaSeqMetrics \
      I=input.bam \
      O=output.RNA_Metrics \
      REF_FLAT=ref_flat.txt \
      STRAND=SECOND_READ_TRANSCRIPTION_STRAND \
      RIBOSOMAL_INTERVALS=ribosomal.interval_list
</code></pre>

<p>I have a couple of bam files that were aligned using mm9 ensembl gtf as annotation, i did a lot of search and found this link Ribosomal Intervals For Collectrnaseqmetrics but this link or any other source does not explain how to produce or find ribo interval list from ensembl annotation</p>

<rna-seq>
ribo interval list for picard ensembl mm9 gtf
# ribo interval list for picard ensembl mm9 gtf

(Asked by: username_0 on 2017-07-19)

Could some one please help me with understanding how to generate the ribosomal interval list that is required to when using picard metrics this step
java -jar picard.jar CollectRnaSeqMetrics \
      I=input.bam \
      O=output.RNA_Metrics \
      REF_FLAT=ref_flat.txt \
      STRAND=SECOND_READ_TRANSCRIPTION_STRAND \
      RIBOSOMAL_INTERVALS=ribosomal.interval_list

I have a couple of bam files that were aligned using mm9 ensembl gtf as annotation, i did a lot of search and found this link Ribosomal Intervals For Collectrnaseqmetrics but this link or any other source does not explain how to produce or find ribo interval list from ensembl annotation

**Tags:** rna-seq

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## 1 answers

### Answer by username_1 on 2017-07-19. Score: 3 | Accepted answer

At least with Ensembl annotations, you can grep rRNA foo.gtf > rRNA.gtf to get the annotated rRNAs. Note however, that the 45S cassette isn't in the mouse reference genome, so you'll never have alignments to 18S, 5.8S, or 28S rRNA. I always get the feeling that the Collect*Metrics programs are made with human sequences in mind, since the cassette is included in the human reference genome.
Anyway, while you can use the annotation and reference sequence to make picard happy, make sure to append the Rn45S sequence to your genome if you want to actually measure rRNA levels. In my experience this is only useful for rRNA-depleted samples, since the rates can vary wildly there.
Edit: To make a file compatible with picard's "interval list", one can do the following with the GTF file:
grep rRNA foo.gtf | awk 'BEGIN{OFS="\t"}{print $1, $4-1, $5}' > foo.bed

According to the picard documentation a BED file can be used as input and the above awk code extracts the appropriate columns and switches to 0-based coordinates used in the BED format.

> **Comments:**
> **username_0**: Interesting, can I directly use the output of `grep rRNA foo.gtf > rRNA.gtf` as input file? or is there any further modification necessary? I did not understand when you say `make picard happy` I am using mm9 reference and yes the sequencing done was using ribo depleted technique (could you please point towards some literature or steps)  
> **username_1**: @novicebioinforesearcher I've updated my reply with the conversion to BED, which allegedly should work. "make picard happy" is another way of saying "give picard a file it can use as input". The results would still not be terribly useful without adding in the Rn45S sequence.  
> **username_0**: I never came across Rn45S concept (thanks for pointing it out) and have not seen any literature in the tools that use such list what would be your thoughts as to why they would not be speak about it?  
> **username_1**: @novicebioinforesearcher Many reference genomes (in particular, human and fly) contain it already, so there'd be no need to use Rn45S there. I don't know why it was excluded from the mouse genome, it's one of the things that's always bothered me.  

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