Close Encounters of the Third Kind; Circumventing the Effects of “Extraterrestrial” DNA

Scott Tighe, Chris Mason, and the Metagenomics Microbiome Research Group (MMRG)

As the field of metagenomics and microbiome continue to growth at an unprecedented rate, the need for proper controls is paramount to ascertain “truth”. Property designed microbiome experiments should include a minimum of three types of controls; positive controls, and two types of negative controls;  trip blank negatives and laboratory processing controls and, as with all scientific experiments,  more controls can help pinpoint all system bias which ultimately contribute to valid results. In recent years, a variety of gDNA microbial reference controls have been developed, but deficits still remain, including proper intact whole cell microbial standards to  account for DNA extraction bias as well as certified DNA-free reagents to support a “perfect” negative control. Since negative controls are critical, and must be included with all experiments, they should include samples such as trip blanks, reagents blanks, process blanks, which all help control for the “extraterrestrial” DNA found in samples that are unrelated to the sample itself also known as the “Kitome”. The “Kitome” is the contaminating DNA of kits and reagents used to extract the DNA and synthesize the library.  These DNA contribute to the final results and must be circumvented to obtain proper results.