checks for spike-in sequence controls 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?
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:


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