Fecal sample collection methods and time of day impact microbiome composition and short chain fatty acid concentrations

Abstract

Associations between the human gut microbiome and health outcomes continues to be of great interest, although fecal sample collection methods which impact microbiome studies are sometimes neglected. Here, we expand on previous work in sample optimization, to promote high quality microbiome data. To compare fecal sample collection methods, amplicons from the bacterial 16S rRNA gene (V4) and fungal (ITS2) region, as well as short chain fatty acid (SCFA) concentrations were determined in fecal material over three timepoints. We demonstrated that spot sampling of stool results in variable detection of some microbial members, and inconsistent levels of SCFA; therefore, sample homogenization prior to subsequent analysis or subsampling is recommended. We also identify a trend in microbial and metabolite composition that shifts over two consecutive stool collections less than 25 h apart. Lastly, we show significant differences in bacterial composition that result from collecting stool samples in OMNIgene·Gut tube (DNA Genotec) or Stool Nucleic Acid Collection and Preservation Tube (NORGEN) compared to immediate freezing. To assist with planning fecal sample collection and storage procedures for microbiome investigations with multiple analyses, we recommend participants to collect the first full bowel movement of the day and freeze the sample immediately after collection.

Advancing human gut microbiota research by considering gut transit time

“…18 81 91 92 In a recent publication, faecal SCFA concentrations and microbial diversity clustered according to stool consistency assessed by the BSS with higher levels of SCFA detected in looser stools, reflecting a shorter colonic transit. 93 Furthermore, increased CTT has also been associated with increased proteolytic fermentation in the colon in both healthy subjects 10 and patients with Parkinson's disease. 94 In Parkinson's disease, constipation and long CTT are common complications.…”

“…Long CTT has been associated with reduced faecal SCFA indicating either increased absorption, lower availability of fermentable polysaccharides in the colon, and/or changed activity 18 81 91 92. In a recent publication, faecal SCFA concentrations and microbial diversity clustered according to stool consistency assessed by the BSS with higher levels of SCFA detected in looser stools, reflecting a shorter colonic transit 93…”

Changes to the Gut Microbiome in Young Children Showing Early Behavioral Signs of Autism

“…Extraction controls were also processed following the same protocol as frozen stool samples. Fecal samples for SCFA analysis were frozen at −80 • C immediately after homogenization, and then transferred on dry ice to the Science Analytical Facility at Edith Cowan University, Western Australia for SCFA quantification as previously described (Jones et al, 2021).…”

“…Bacterial DNA and gut microbiome mock community (https:// www.atcc.org/products/msa-1006) were amplified using 16S primers 515F (Turner et al, 1999) and 806R (Caporaso et al, 2011), while fungal DNA was amplified using ITS2 primers FSeq and RSeq (Heisel et al, 2015), each with a 6-8 bp unique barcode. The PCR reactions, library preparation, and sequencing were performed according to methods previously described (Jones et al, 2021).…”

“…Sequences were demultiplex using unique molecular barcodes with no mismatches allowed before they were removed along with primer sequences using Cutadapt (Martin, 2011). Quality filtering using DADA2 (Callahan et al, 2016) was performed as previously described (Jones et al, 2021). The Genome Taxonomy reference database (Version 202) was formatted for use with DADA2 (https://zenodo.org/record/4735821#.YN18Om4RWis), and the UNITE general FASTA release for fungi Version 8.3 (Kõljalg et al, 2005) were used to classify 16S and ITS2 sequence variants, respectively, each with a minimum of 80% bootstrap.…”