Reliable dietary data is essential for clinicians and practitioners to make appropriate recommendations. However, current tracking methods often rely on self-reporting and a person's ability to accurately record what they eat. These methods are often prone to errors in memory, bias associated with human memory, and misreporting errors. The data from this type of reporting method often overestimates the importance of rare food items and underestimates common ones.

Therefore, the researchers determined there is a need for alternative methods of quantifying human diet composition. DNA metabarcoding is an alternative way to obtain dietary information that uses food DNA in stool as a biomarker. "I think of DNA metabarcoding very much like a barcode at a supermarket. We can think of a particular DNA sequence as a unique identifier for a particular food species," said second study author Brianna Petrone, a graduate student at Duke University School of Medicine.

To conduct their study, the researchers pulled DNA out of cold storage that had been extracted from stool samples from a previous study. "We happened to do a study a couple years ago where we were preparing foods for participants in a microbiome diet intervention, and we knew exactly what they were eating in a given week when their stool was being collected," said Lawrence David, PhD, assistant professor, Center for Genomic and Computational Biology, Duke Molecular Genetics and Microbiology.

The researchers sequenced a barcode region from chloroplast DNA (the P6 loop of the trnL [UAA] intron) in stool samples from 11 individuals consuming both controlled and freely selected diets. In total, they observed a PCR band in 50% of the samples from the prepared-diet study. The obtained over 2 million sequence reads that perfectly matched 78 sequences from the reference database. The majority of sequenced plant DNA matched common human food plants, including grains, vegetables, fruits and herbs.

The relatively high PCR failure rate and inability to distinguish some dietary plants at the sequence level suggest the potential for future refinements to improve the method. For example, cabbage, broccoli, Brussel sprouts, and kohlrabi are all cultivars of the same species, and researchers were unable to tell them apart by their sequence in the chloroplast barcode region. Coffee was the only food recorded in the diet that was never detected with DNA metabarcoding, perhaps because its DNA was deteriorated or diluted by roasting and brewing. Improvements to DNA metabarcoding are occurring rapidly. Many technical challenges are being overcome including optimizing sample handling and extraction and developing computer algorithms that can more effectively detect and remove aberrant DNA sequences.

"Similar to this study, I could imagine this getting used on archived DNA to see whether or not there are underlying dietary differences that might explain some of the microbiome patterns that may have been observed in a study," said David. "Going forward, we can also imagine this being used in new microbiome studies to identify relationships between specific foods and gut bacteria, as well as in broader studies of nutrition as a complement to traditional diet assessment techniques."

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