Answers.Body: <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>
  
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-09-04T14:28:59.853Z
Answers.Text: Does Anaquin/Sequin allow for the design of new spike-in controls that don't align to a set of references?
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Answers.CreationDate: 2017-09-04T15:08:14.140Z
Answers.Text: @719016 Sequins assume the spiked-in reads aligned to a synthetic reference.
Answers.UserDisplayName: SmallChess
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-09-04T16:09:59.113Z
Answers.Text: Does it allow for the design of new spikeins wrt a new synthetic reference?
Answers.UserDisplayName: 719016
Answers.ContentLicense: CC BY-SA 3.0
Answers.CreationDate: 2017-09-04T16:11:13.450Z
Answers.Text: Does it check if the set of synthetic reference is unique against other Genomes and unique against itself?
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Answers.UserDisplayName: SmallChess
Body: <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>
  
Tags: <ngs><illumina><errors><spike-in>
Title: checks for spike-in sequence controls
ThreadText: # 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**: @username_0 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?  
  
  ---
  
  