<p><strong>Disclaimer</strong>: I'm a developer for <a href="http://www.sequin.xyz" rel="nofollow noreferrer">http://www.sequin.xyz</a>.</p>

<p>Sequin is a new set of spiked-in controls for next-generation sequencing, and that includes Illumina. We design controls for RNA-Seq, genome sequencing, metagenomics etc.</p>

<p>Please study our <a href="http://www.sequin.xyz/about/papers/" rel="nofollow noreferrer">papers</a> if you want more details.</p>

<ul>
<li><strong>Reference Standards For Next-Generation Sequencing</strong> by Hardwick should be a good start. </li>
</ul>

<p>Here's my list:</p>

<ol>
<li>Measured counts against input concentration. Pearson's correlation, spearman correlation and regression slope.</li>
<li>PCA analysis</li>
<li>Whether the synthetic aligned to the synthetic reference</li>
<li>Spiked-in amount (e.g. dilution)</li>
<li>Diagnostic statistics</li>
<li>ROC Curve</li>
<li>Detection limit</li>
</ol>

<p>(1) is important because the controls should give you correlation = 1, and slope = 1 in a perfect flawless experiment. This is a sample output (you can read more about it in our papers):</p>

<p><a href="https://i.sstatic.net/DjqEV.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/DjqEV.png" alt="enter image description here"></a></p>

<p>(2) This is our sample PCA (our paper has the details)</p>

<p><a href="https://i.sstatic.net/vGTh0.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/vGTh0.png" alt="enter image description here"></a></p>

<p>(3) is also important but you can only do that in simulations.</p>

<p>(5)</p>

<p><a href="https://i.sstatic.net/zaEfm.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/zaEfm.png" alt="enter image description here"></a> </p>

<p>(6)</p>

<p><a href="https://i.sstatic.net/cg6ss.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/cg6ss.png" alt="enter image description here"></a></p>

<p>(7)</p>

<p><a href="https://i.sstatic.net/NRF6p.png" rel="nofollow noreferrer"><img src="https://i.sstatic.net/NRF6p.png" alt="enter image description here"></a></p>

CC BY-SA 3.0
2017-09-04T14:28:59.853Z
Does Anaquin/Sequin allow for the design of new spike-in controls that don't align to a set of references?
719016
CC BY-SA 3.0
2017-09-04T15:08:14.140Z
@719016 Sequins assume the spiked-in reads aligned to a synthetic reference.
SmallChess
CC BY-SA 3.0
2017-09-04T16:09:59.113Z
Does it allow for the design of new spikeins wrt a new synthetic reference?
719016
CC BY-SA 3.0
2017-09-04T16:11:13.450Z
Does it check if the set of synthetic reference is unique against other Genomes and unique against itself?
719016
CC BY-SA 3.0
2017-09-04T06:04:41.237Z
2017-09-04T06:09:45.713Z
2017-09-04T06:09:45.713Z
SmallChess
<p>I would like to know what do people verify when designing/using spike-in controls, to be used in sequencing experiments (mainly Illumina). So far I came up with this list:</p>

<ul>
<li>Does it align only to a given genome synthetic reference?</li>
<li>Does it contain G-quadruplexes? E.g. <a href="http://bioinformatics.ramapo.edu/QGRS/index.php" rel="nofollow noreferrer">QGRS Mapper</a></li>
<li>Does it contain known Illumina error motifs? (Do this matter anymore for recent platforms/chemistries?) See <a href="https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-0976-y" rel="nofollow noreferrer">this article</a></li>
<li>Does it contain other motifs known to be polymerase inhibitors? <a href="https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923904/" rel="nofollow noreferrer">Ref</a></li>
</ul>

<p>Anything else?</p>

<ngs><illumina><errors><spike-in>
checks for spike-in sequence controls
# checks for spike-in sequence controls

(Asked by: username_0 on 2017-08-31)

I would like to know what do people verify when designing/using spike-in controls, to be used in sequencing experiments (mainly Illumina). So far I came up with this list:

* Does it align only to a given genome synthetic reference?
* Does it contain G-quadruplexes? E.g. [QGRS Mapper](http://bioinformatics.ramapo.edu/QGRS/index.php)
* Does it contain known Illumina error motifs? (Do this matter anymore for recent platforms/chemistries?) See [this article](https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-0976-y)
* Does it contain other motifs known to be polymerase inhibitors? [Ref](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4923904/)

Anything else?

**Tags:** ngs, illumina, errors, spike-in

---

## 1 answers

### Answer by username_1 on 2017-09-04. Score: 4 | Accepted answer

**Disclaimer**: I'm a developer for <http://www.sequin.xyz>.

Sequin is a new set of spiked-in controls for next-generation sequencing, and that includes Illumina. We design controls for RNA-Seq, genome sequencing, metagenomics etc.

Please study our [papers](http://www.sequin.xyz/about/papers/) if you want more details.

* **Reference Standards For Next-Generation Sequencing** by Hardwick should be a good start.

Here's my list:

1. Measured counts against input concentration. Pearson's correlation, spearman correlation and regression slope.
2. PCA analysis
3. Whether the synthetic aligned to the synthetic reference
4. Spiked-in amount (e.g. dilution)
5. Diagnostic statistics
6. ROC Curve
7. Detection limit

(1) is important because the controls should give you correlation = 1, and slope = 1 in a perfect flawless experiment. This is a sample output (you can read more about it in our papers):

[![enter image description here](https://i.sstatic.net/DjqEV.png)](https://i.sstatic.net/DjqEV.png)

(2) This is our sample PCA (our paper has the details)

[![enter image description here](https://i.sstatic.net/vGTh0.png)](https://i.sstatic.net/vGTh0.png)

(3) is also important but you can only do that in simulations.

(5)

[![enter image description here](https://i.sstatic.net/zaEfm.png)](https://i.sstatic.net/zaEfm.png)

(6)

[![enter image description here](https://i.sstatic.net/cg6ss.png)](https://i.sstatic.net/cg6ss.png)

(7)

[![enter image description here](https://i.sstatic.net/NRF6p.png)](https://i.sstatic.net/NRF6p.png)

> **Comments:**
> **username_0**: Does Anaquin/Sequin allow for the design of new spike-in controls that don't align to a set of references?  
> **username_1**: @719016 Sequins assume the spiked-in reads aligned to a synthetic reference.  
> **username_0**: Does it allow for the design of new spikeins wrt a new synthetic reference?  
> **username_0**: Does it check if the set of synthetic reference is unique against other Genomes and unique against itself?  

---

