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adrien.andre 03-29-2016 01:00 AM

subsampling before/after contaminant removing?
I've got some 16S sequences obtained with a miseq sequencer.
I have sequenced several negative control samples to identify contaminants that I have to remove them from my actual samples.
I was thinking about deleting the contaminated OTU from each sample if its number of reads is not present 10x more than in the highest contaminated negative control.
in short, if I have this pattern for OTU x :
sample A : 20 reads
Sample B : 300 reads
Negative control C : 3 reads
Negative control D : 12 reads

I will delete this OTU for sample A but not for sample B as 300> 120

This being said, here is my question :
When should the sub-sampling being performed? should it be before or after removing the contaminants?

I was first thinking about subsampling after the contaminant removing step.
but then, I thought that the rule for removing contaminant stated previously should be efficient only in a subsampled dataset.

therefore, should i rather subsample before removing the contaminants? But then, the problem is that some samples are way more contaminated than others, which means that the resulting sequencing depth will vary from a sample to another...

Or should I subsample twice? before AND after? :confused:

Thank you in advance for your precious advices :)


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