Batch effects account for the main findings of an in utero human intestinal bacterial colonization study.

A recent study by Rackaityte et al. reported evidence for a low level of bacterial colonization, specifically of Micrococcus luteus, in the intestine of second trimester human fetuses. We have re-analyzed their sequence data and identified a batch effect which violates the underlying assumptions of the bioinformatic method used for contamination removal. This batch effect resulted in Micrococcus not being identified as a contaminant in the original work and being falsely assigned to the fetal samples. We further provide evidence that the micrographs presented by Rackaityte et al. are unlikely to show Micrococci or other bacteria as the size of the particles shown exceeds that of related bacterial cells. Finally, phylogenetic analysis showed that the microbes cultured from the fetal samples differed significantly from those detected by sequencing. Overall, our findings show that the presence of Micrococcus in the fetal gut is not supported by the primary sequence data. Our findings underline important aspects of the nature of contamination for both sequencing and culture approaches in microbiome studies and the appropriate use of automated contamination identification tools.