Using DNA Metabarcoding To Evaluate the Plant Component of Human Diets: a Proof of Concept.

Dietary intake is difficult to measure reliably in humans because approaches typically rely on self-reporting, which can be incomplete and biased. In field studies of animals, DNA sequencing-based approaches such as metabarcoding have been developed to characterize diets, but such approaches have not previously been widely applied to humans. Here, we present data derived from sequencing of a chloroplast DNA marker (the P6 loop of the trnL [UAA] intron) in stool samples collected from 11 individuals consuming both controlled and freely selected diets. The DNA metabarcoding strategy resulted in successful PCR amplification in about 50% of samples, which increased to a 70% success rate in samples from individuals eating a controlled plant-rich diet. Detection of plant taxa among sequenced samples yielded a recall of 0.86 and a precision of 0.55 compared to a written diet record during controlled feeding of plant-based foods. The majority of sequenced plant DNA matched common human food plants, including grains, vegetables, fruits, and herbs prepared both cooked and uncooked. Moreover, DNA metabarcoding data were sufficient to distinguish between baseline and treatment diet arms of the study. Still, the relatively high PCR failure rate and an inability to distinguish some dietary plants at the sequence level using the trnL-P6 marker suggest that future methodological refinements are necessary. Overall, our results suggest that DNA metabarcoding provides a promising new method for tracking human plant intake and that similar approaches could be used to characterize the animal and fungal components of our omnivorous diets.IMPORTANCE Current methods for capturing human dietary patterns typically rely on individual recall and as such are subject to the limitations of human memory. DNA sequencing-based approaches, frequently used for profiling nonhuman diets, do not suffer from the same limitations. Here, we used metabarcoding to broadly characterize the plant portion of human diets for the first time. The majority of sequences corresponded to known human foods, including all but one foodstuff included in an experimental plant-rich diet. Metabarcoding could distinguish between experimental diets and matched individual diet records from controlled settings with high accuracy. Because this method is independent of survey language and timing, it could also be applied to geographically and culturally disparate human populations, as well as in retrospective studies involving banked human stool

Using DNA Metabarcoding To Evaluate the Plant Component of Human Diets: a Proof of Concept.

Dietary intake is difficult to measure reliably in humans because approaches typically rely on self-reporting, which can be incomplete and biased. In field studies of animals, DNA sequencing-based approaches such as metabarcoding have been developed to characterize diets, but such approaches have not previously been widely applied to humans. Here, we present data derived from sequencing of a chloroplast DNA marker (the P6 loop of the trnL [UAA] intron) in stool samples collected from 11 individuals consuming both controlled and freely selected diets. The DNA metabarcoding strategy resulted in successful PCR amplification in about 50% of samples, which increased to a 70% success rate in samples from individuals eating a controlled plant-rich diet. Detection of plant taxa among sequenced samples yielded a recall of 0.86 and a precision of 0.55 compared to a written diet record during controlled feeding of plant-based foods. The majority of sequenced plant DNA matched common human food plants, including grains, vegetables, fruits, and herbs prepared both cooked and uncooked. Moreover, DNA metabarcoding data were sufficient to distinguish between baseline and treatment diet arms of the study. Still, the relatively high PCR failure rate and an inability to distinguish some dietary plants at the sequence level using the trnL-P6 marker suggest that future methodological refinements are necessary. Overall, our results suggest that DNA metabarcoding provides a promising new method for tracking human plant intake and that similar approaches could be used to characterize the animal and fungal components of our omnivorous diets.IMPORTANCE Current methods for capturing human dietary patterns typically rely on individual recall and as such are subject to the limitations of human memory. DNA sequencing-based approaches, frequently used for profiling nonhuman diets, do not suffer from the same limitations. Here, we used metabarcoding to broadly characterize the plant portion of human diets for the first time. The majority of sequences corresponded to known human foods, including all but one foodstuff included in an experimental plant-rich diet. Metabarcoding could distinguish between experimental diets and matched individual diet records from controlled settings with high accuracy. Because this method is independent of survey language and timing, it could also be applied to geographically and culturally disparate human populations, as well as in retrospective studies involving banked human stool

FRUIT, a scar-free system for targeted chromosomal mutagenesis, epitope tagging, and promoter replacement in Escherichia coli and Salmonella enterica.

Recombineering is a widely-used approach to delete genes, introduce insertions and point mutations, and introduce epitope tags into bacterial chromosomes. Many recombineering methods have been described, for a wide range of bacterial species. These methods are often limited by (i) low efficiency, and/or (ii) introduction of "scar" DNA into the chromosome. Here, we describe a rapid, efficient, PCR-based recombineering method, FRUIT, that can be used to introduce scar-free point mutations, deletions, epitope tags, and promoters into the genomes of enteric bacteria. The efficiency of FRUIT is far higher than that of the most widely-used recombineering method for Escherichia coli. We have used FRUIT to introduce point mutations and epitope tags into the chromosomes of E. coli K-12, Enterotoxigenic E. coli, and Salmonella enterica. We have also used FRUIT to introduce constitutive and inducible promoters into the chromosome of E. coli K-12. Thus, FRUIT is a versatile, efficient recombineering approach that can be applied in multiple species of enteric bacteria

Comparison of FRUIT to pKD13-mediated recombineering.

a<p>Frequency with which candidate colonies were successfully verified.</p>b<p>Number of colonies/number of viable cells.</p>c<p>Relative efficiency of FRUIT as compared to pKD13.</p

Schematic of FRUIT method.

<p>(A) Schematic of FRUIT for introducing point mutations or deletions. PCR product is amplified from the recombineering template plasmid (pAMD001), incorporating flanking sequence with identity to the desired site of recombination. This PCR product is introduced into cells expressing λ recombinase proteins and recombinants are selected using the <i>thyA</i> marker (growth on media lacking thymine). A mutation can then be introduced by recombineering a second PCR product, selecting for recombinants using counter-selection of <i>thyA</i> (growth in the presence of trimethoprim). (B) Schematic of FRUIT for introducing FLAG tags. As above, except that loss of <i>thyA</i> occurs spontaneously due to homologous recombination of duplicate sets of FLAG tags.</p

FRUIT promoter swaps in MG1655 (<i>E. coli</i> K-12).

<p>(A) Schematic indicating the plasmid templates used for FRUIT. (B) Schematic indicating replacement of the <i>lacZYA</i> promoter with P_high, P_med, P_low or P_rha promoters. (C) β-galactosidase assay in Δ<i>lacZ</i> MG1655 and mutant strains with P_high, P_med or P_low driving expression of <i>lacZYA</i> (cells were grown without IPTG). (D) β-galactosidase assay in Δ<i>lacZ</i> MG1655 and a mutant strain with P_rha driving expression of <i>lacZYA</i>. Assays were performed ± rhamnose.</p

List of strains and plasmids.

<p>List of strains and plasmids.</p