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Figure 4

Posted by DotBlot on 02 Nov 2012 at 21:25 GMT

Please help me to understand this figure #4.
I am new to 16S rRNA sequencing and would like to see if I understand what is going on here. In the methods, you are saying that you mixed these genomic DNAs in equal amounts relative to 16rRNA abundance. So theoretically after sequencing there should be ruffly 5% of the reads associated with each organism but in fact there is huge variablity and that this is associated with the primer design.. Specifically the forward primer. Shouldn't this be a major concern to the microbiome investigations?
Thanks for your response. Peace.

No competing interests declared.

RE: Figure 4

pschloss replied to DotBlot on 06 Nov 2012 at 19:56 GMT

Well, yes and no. We really didn't want this figure to get at bias of the primers since the study wasn't designed to do that. My understanding is that there was a miscalculation in the DNA concentrations and pooling that threw off their relative abundances. So we're pretty sure they weren't even going into the tube. But you are correct, that there are problems with primer design and potentially PCR bias, in general. If one wanted to test this, they would need to create replicates of the mock and sequence those replicates. Then they would need to use qPCR to quantify each population in the pool to make sure we knew what % went in. Like I said, this isn't how our study was designed.

No competing interests declared.