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  • subsampling before/after contaminant removing?

    Hi,
    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?

    Thank you in advance for your precious advices

    Adrien

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