<p>I actually figured out what was going on some time ago:</p>
<p>The over-represented sequence is the <a href="https://teichlab.github.io/scg_lib_structs/methods_html/10xChromium3.html" rel="nofollow noreferrer">template switching oligo (TSO)</a> that is used for the synthesis of the second strand (the example is for the v2 chemistry but the principle is the same for v3 and later).<br />
This is a simplified illustration from the 10x manual:<br />
<a href="https://i.sstatic.net/2936b.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/2936b.png" alt="template switching and second strand synthesis" /></a></p>
<p>As you can see the cDNA insert should be between the polyA capture probe and the TSO. In the second sample there are many cases with no insert and the TSO is followed by the poly A sequence. Actually the TSO should not be present in the final read after addition of Illumina adapters. But without any insert the fragmentation does not work and many reads still have this technical sequence.<br />
So @merv was correct. Many reads contain no insert.</p>
<h2>Why did this happen?</h2>
<p>Why were there many reads without insert in this experiment and why are some samples more heavily affected than others? As I mentioned, this were eosinophil granulozytes and these fellas contain many proteases, DNAses and RNAses to destroy invading pathogens. Unfortunately, they also destroy mRNA in single cell experiments.<br />
Interestingly, the eosinophily in the second sample received an activating treatment and probably contained even more or more active RNAses.</p>

CC BY-SA 4.0
2021-06-28T13:48:20.520Z
2021-06-28T13:48:20.520Z
PPK
<p><em>Answer from @merv, converted from comment:</em></p>
<p>Perhaps it's an insert size issue. Can you get the post library construction QC info? You might also see this in the BAMs, with the first showing a full bell-shaped peaks, and the latter truncating the peaks at the cleavage site. (Most 10X datasets I've seen have very few reads traversing the 3' end cleavage site.)</p>

CC BY-SA 4.0
2021-06-25T11:12:54.260Z
2021-06-25T11:12:54.260Z
gringer
<p>I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality.<br />
According to the Cell Ranger summary 56% of the reads can be mapped to the transcriptome in the first sample and only 32% in the second sample. Also the number of cells identified in Cell Ranger are very different (5000 for sample 1 and 600 for sample 2).</p>
<p>When I look at the raw data in FastQC I see that the second sample has a lot of poly-A sequences in the R2 read that obviously can't be mapped to the transcriptome.</p>
<p>Sample 1:<br />
<a href="https://i.sstatic.net/ElEFk.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/ElEFk.png" alt="per base sequence content sample 1" /></a></p>
<p>Sample 2:
<a href="https://i.sstatic.net/nogKK.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/nogKK.png" alt="per base sequence content sample 2" /></a></p>
<p>Also in many cases there is the following sequence in front of the poly-A:<br />
<code>AAGCAGTGGTATCAACGCAGAGTACATGGG</code></p>
<p>To me it looks as if most of the reads do not contain an insert and are primer dimers. Is this connected to the lower cell count in the second sample?<br />
Maybe some of you have already seen something like this and have a suggestion on how to avoid such a problem in the future.</p>
<p>Thanks a lot</p>

CC BY-SA 4.0
2021-06-25T22:53:46.760Z
Hi @PPK, could you please add your solution as an *answer* to this question and "accept" it? That will help others who encounter the same problem, and stop the StackExchange Community bot from bumping this question from time to time to fish for more answers.
gringer
<scrnaseq><ngs><10x-genomics>
High percentage of poly A sequences in 10X chromium R2 read
# High percentage of poly A sequences in 10X chromium R2 read

(Asked by: username_0 on 2019-08-09)

I'm currently analyzing two samples of eosinophil cells isolated from mouse lung and the samples are of very different quality.  
According to the Cell Ranger summary 56% of the reads can be mapped to the transcriptome in the first sample and only 32% in the second sample. Also the number of cells identified in Cell Ranger are very different (5000 for sample 1 and 600 for sample 2).

When I look at the raw data in FastQC I see that the second sample has a lot of poly-A sequences in the R2 read that obviously can't be mapped to the transcriptome.

Sample 1:  
[![per base sequence content sample 1](https://i.sstatic.net/ElEFk.png)](https://i.sstatic.net/ElEFk.png)

Sample 2:
[![per base sequence content sample 2](https://i.sstatic.net/nogKK.png)](https://i.sstatic.net/nogKK.png)

Also in many cases there is the following sequence in front of the poly-A:  
`AAGCAGTGGTATCAACGCAGAGTACATGGG`

To me it looks as if most of the reads do not contain an insert and are primer dimers. Is this connected to the lower cell count in the second sample?  
Maybe some of you have already seen something like this and have a suggestion on how to avoid such a problem in the future.

Thanks a lot

**Tags:** scrnaseq, ngs, 10x-genomics
> **Comments:**
> **username_1**: Hi @PPK, could you please add your solution as an *answer* to this question and "accept" it? That will help others who encounter the same problem, and stop the StackExchange Community bot from bumping this question from time to time to fish for more answers.  

---

## 2 answers

### Answer by username_0 on 2021-06-28. Score: -1 | Accepted answer

I actually figured out what was going on some time ago:

The over-represented sequence is the [template switching oligo (TSO)](https://teichlab.github.io/scg_lib_structs/methods_html/10xChromium3.html) that is used for the synthesis of the second strand (the example is for the v2 chemistry but the principle is the same for v3 and later).  
This is a simplified illustration from the 10x manual:  
[![template switching and second strand synthesis](https://i.sstatic.net/2936b.png)](https://i.sstatic.net/2936b.png)

As you can see the cDNA insert should be between the polyA capture probe and the TSO. In the second sample there are many cases with no insert and the TSO is followed by the poly A sequence. Actually the TSO should not be present in the final read after addition of Illumina adapters. But without any insert the fragmentation does not work and many reads still have this technical sequence.  
So @merv was correct. Many reads contain no insert.

## Why did this happen?

Why were there many reads without insert in this experiment and why are some samples more heavily affected than others? As I mentioned, this were eosinophil granulozytes and these fellas contain many proteases, DNAses and RNAses to destroy invading pathogens. Unfortunately, they also destroy mRNA in single cell experiments.  
Interestingly, the eosinophily in the second sample received an activating treatment and probably contained even more or more active RNAses.


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### Answer by username_1 on 2021-06-25. Score: 1

*Answer from @merv, converted from comment:*

Perhaps it's an insert size issue. Can you get the post library construction QC info? You might also see this in the BAMs, with the first showing a full bell-shaped peaks, and the latter truncating the peaks at the cleavage site. (Most 10X datasets I've seen have very few reads traversing the 3' end cleavage site.)


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