Abstract
Stool contains DNA shed from cells of the gastrointestinal (GI) tract and has great potential as a bio-specimen for non-invasive, nucleic acid-based detection of GI diseases. Whereas methods for studying fecal microbiome DNA are plentiful, there is a lack of well-characterized procedures for stabilization, isolation, and quantitative analysis of fecal host DNA. We report an optimized pipeline for fecal host DNA analysis from the point-of-collection to droplet digital PCR (ddPCR) absolute quantification of host-specific gene targets. We evaluated multiple methods for preservation and isolation of host DNA from stool to identify the highest performing methods. To quantify host DNA, we developed sensitive, human-specific ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimized methods to perform absolute quantification of short (<100 bp) host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalized patients. These specimens allowed assessment of host DNA day-to-day variability in stool specimens with widely varying physical characteristics (i.e., Bristol scores). We further extended this approach to mouse stool analysis, to enable fecal host DNA studies in animal disease models as well.
Footnotes
↵* mtewari{at}med.umich.edu