Positive control negative control8/7/2023 ![]() Common to all these publications is the lack of controls, paired with the investigation of relatively low biomass microbiomes like mucosa (Zuo et al. During our research we also still noticed several high impact publications, which report results which are potentially indistinguishable from contaminations. ‘appropriate controls were used at all steps’, without further details or ‘methods have previously been validated with positive controls’). when samples were also used for qPCR), or the descriptions were insufficient to judge whether controls were adequate (e.g. In some of these cases, however, it was unclear if the negative controls were actually sequenced (e.g. From the 265 publications utilizing high-throughput community sequencing of any type (16S, metagenomics, metatranscriptomics, 18S, ITS, virome, PacBio, Nanopore, others), only 30% (79) reported using any type of negative control, and only 10% (27) reported using a positive control. To clarify the extent of this, we reviewed all publications from the 2018 issues of Microbiome and the ISME journal (manually, as well as keyword searches for ‘mock’, ‘blank’ and ‘control’). In microbiome research, however, controls have not been included in the majority of published studies. It is good practice to perform experiments with controls, to ensure that all procedures were correctly performed, and that none of the steps have introduced false positive or false negative results. One particular problem exists, which has been overlooked for a long time: the lack of controls in microbiome research. There are various other concerns which are not specific to this field, but also apply here, like the crisis in reproducibility (Schloss 2018), or the public availability of data in a FAIR (Findable, Accessible, Interoperable and Reusable) way (Langille, Ravel and Fricke 2018). 2016), among many others), which makes their interpretation difficult. It is also clear that these various DNA extraction methods might not produce very overlapping results (e.g. Most researchers are aware of the tremendous impact that DNA extraction has on the outcome of any microbiome study (Costea et al. The use of shotgun sequencing or targeted DNA amplicon sequencing to characterize the microbiome has its complications though. ![]() The subsequent rise in interest in this field was tremendous, leading to numerous publications in highly respected journals, e.g. It was, and still is, exciting to sequence the microbial community of an environment to its full extent, and to learn about all its inhabitants and their possible functions. The hallmark publication by (Venter et al. The microbiome field is a relatively new field of research. ![]()
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