<p>We've found ribosomal RNA to be less of a problem with sequencing that depends on polyA, which suggests the issue might be in the library preparation, rather than the selection.</p>

<p>Many polyA RNA library preparation methods involve amplification, rather than selection, which means that existing transcripts that are present in very high abundance (such as rRNA) will still show up in the mixture.</p>

<p>But polyA-tailed RNA isn't the only RNA present in a sample. For a comprehensive look at RNA, the whole-RNA sample preparation methods can't use <em>any</em> form of selection to identify target molecules. In this case, it's typically recommended to use a form of ribosomal RNA removal, where a probe to ribosomal RNA is attached to magnetic beads (or a column, or similar), and used to fish out out ribosomal RNA, leaving the remainder behind. Unfortunately those probes don't work perfectly, either due to the ribosomal RNA being damaged at the probe site, or the probe not matching the ribosomal RNA molecule, or the RNA just not getting into the right physical state to bind with the probe. All these things mean incomplete removal of ribosomal RNA, ending up with a situation where the highly-abundant ribosomal RNA still pokes through in the reads.</p>

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2018-02-17T02:15:21.587Z
2018-02-18T18:04:16.227Z
2018-02-18T18:04:16.227Z
gringer
<p>In <a href="https://bioinformatics.stackexchange.com/a/3194/1771">this answer</a> (actually in a comment), it is stated that:</p>

<blockquote>
  <p>As you've noticed from your own analysis, the ribosomal genes have quite variable expression across cells. They're expressed everywhere, and quite <strong>difficult to completely remove from a sample (even with polyA selection)</strong>, with success of removal depending on things like the amount of mechanical damage that the RNA has been exposed to.</p>
</blockquote>

<p>Which are the most relevant issues that make Poly(A) selection fail? Looking online, Poly(A) <a href="https://www.researchgate.net/post/For_RNAseq_analysis_what_would_you_advise_poly-A_selection_or_rRNA_depletion2" rel="nofollow noreferrer">is suggested</a> often but I didn't find an explanation for the different ways in which it can fail.</p>

<rna-seq><scrnaseq><rna><ribosomal>
Why is ribosomal RNA difficult to remove even with Poly(A) selection?
# Why is ribosomal RNA difficult to remove even with Poly(A) selection?

(Asked by: username_0 on 2018-02-16)

In [this answer](https://bioinformatics.stackexchange.com/a/3194/1771) (actually in a comment), it is stated that:

> As you've noticed from your own analysis, the ribosomal genes have quite variable expression across cells. They're expressed everywhere, and quite **difficult to completely remove from a sample (even with polyA selection)**, with success of removal depending on things like the amount of mechanical damage that the RNA has been exposed to.

Which are the most relevant issues that make Poly(A) selection fail? Looking online, Poly(A) [is suggested](https://www.researchgate.net/post/For_RNAseq_analysis_what_would_you_advise_poly-A_selection_or_rRNA_depletion2) often but I didn't find an explanation for the different ways in which it can fail.

**Tags:** rna-seq, scrnaseq, rna, ribosomal

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## 1 answers

### Answer by username_1 on 2018-02-17. Score: 5 | Accepted answer

We've found ribosomal RNA to be less of a problem with sequencing that depends on polyA, which suggests the issue might be in the library preparation, rather than the selection.

Many polyA RNA library preparation methods involve amplification, rather than selection, which means that existing transcripts that are present in very high abundance (such as rRNA) will still show up in the mixture.

But polyA-tailed RNA isn't the only RNA present in a sample. For a comprehensive look at RNA, the whole-RNA sample preparation methods can't use *any* form of selection to identify target molecules. In this case, it's typically recommended to use a form of ribosomal RNA removal, where a probe to ribosomal RNA is attached to magnetic beads (or a column, or similar), and used to fish out out ribosomal RNA, leaving the remainder behind. Unfortunately those probes don't work perfectly, either due to the ribosomal RNA being damaged at the probe site, or the probe not matching the ribosomal RNA molecule, or the RNA just not getting into the right physical state to bind with the probe. All these things mean incomplete removal of ribosomal RNA, ending up with a situation where the highly-abundant ribosomal RNA still pokes through in the reads.


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