A Pipeline for Faecal Host DNA Analysis by Absolute Quantification of LINE-1 and Mitochondrial Genomic Elements Using ddPCR

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 faecal microbiome DNA are plentiful, there is a lack of well-characterised procedures for stabilisation, isolation, and quantitative analysis of faecal host DNA. We report an optimised pipeline for faecal 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 even if present in partially degraded form, we developed sensitive, human-specific short-amplicon ddPCR assays targeting repetitive nuclear genomic elements (LINE-1) and mitochondrial genes. We validated the ability of these optimised methods to perform absolute quantification of host DNA in 200 stool DNA extracts from samples that were serially collected from three healthy individuals and three hospitalised 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 faecal host DNA studies in animal disease models as well.

Impact of Preanalytical and Analytical Methods on Cell-Free DNA Diagnostics

“…To assess the viability of cfDNA concentration detection by ddPCR, the total 82 bp LINE-1 cfDNA fragments were quantified on the ddPCR system (Bio-Rad Laboratories), using the modified protocol from He K. et al (2019) .…”

The standardisation of the approach to metagenomic human gut analysis: from sample collection to microbiome profiling

“…) is a mixture of 12 bacterial strains that are typical of the human gut microbiome (Supplementary Table 1). To this sample, approximately 10,000 human PBMCs were added to simulate host cells that are normally present in stool samples 25 . Those samples are hereafter referred to as BL.…”

Technical and Methodological Aspects of Cell-Free Nucleic Acids Analyzes

“…Since stool contains a really large portion of other components that could inhibit downstream analyzes, it is necessary to choose a collection method which allows the maximal recovery of human DNA, preserves human NAs, and inhibits undesirable components. There are some commercial collection devices or storage systems, containing specialized preservatives for the stabilization and protection of NAs in stool samples, available, including the DNA/RNA Shield Fecal Collection tubes (Zymo, Irvine, CA, USA) [ 155 ], the Norgen Stool Nucleic Acid Collection and Preservation Tubes [ 156 , 157 ], or the OMNIgene-GUT buffer (DNA Genotek, Ottawa, ON, Canada) [ 158 ], however, neither of them were found to be dedicated to cfNAs. They generally prevent the growth of many bacteria, fungi, and inactivate viruses, and also offer stabilization of NAs in stool samples from several days up to a few years, most commonly at room temperature, or in a frozen state ( Table S3 ).…”

“…They generally prevent the growth of many bacteria, fungi, and inactivate viruses, and also offer stabilization of NAs in stool samples from several days up to a few years, most commonly at room temperature, or in a frozen state ( Table S3 ). However, it is not yet clear, whether these stabilization solutions are effective for the preservation of the wide range of different DNA length fragments [ 158 ].…”