checks for spike-in sequence controls

Question

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
• Does it contain known Illumina error motifs? (Do this matter anymore for recent platforms/chemistries?) See this article
• Does it contain other motifs known to be polymerase inhibitors? Ref

Anything else?

Answer 1

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 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):

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

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

(5)

(6)

(7)