Sources of Variation in 16S rRNA Based Microbiome Studies: An Inter-Laboratoy Ring Trial. MRI_Ring

Microbiome studies based on data obtained from sequencing amplicons of the 16S rRNA gene are very common but it is known that standardization of the methods for DNA extraction and amplification is lacking. We performed a ring trial between three different laboratories of the Max Rubner-Institut, comparing the technical variation by analyzing aliquots of homogenized stools samples originating from six individual donors. Two different protocols were used for the analysis (based on "NucleoSpin DNA Stool" kit by Macherey-Nagel and "QIAmp Power Fecal DNA" kit) to compare the impact of the DNA extraction on technical variation. In addition all DNA samples were pooled to two libraries that were sequenced on individual Illumina MiSeq machines. Choice of DNA extraction protocol was by far the biggest contributor to technical variation, followed by laboratory, sequencing machine and background variation (i.e. identically treated aliquots).