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Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children.

ABSTRACT: With the emergence of large-scale epidemiologic human microbiome studies, there is a need to understand the reproducibility of microbial DNA sequencing and the impact of specimen collection and processing methods on measures of microbial community composition and structure, with reproducibility studies in infants and young children particularly lacking. Here, we examined batch-to-batch variability and reliability of collection, handling, and processing protocols, testing replicate stool samples from infants and young children using Illumina MiSeq sequencing of the bacterial 16S rRNA gene V4-V5 hypervariable region, evaluating 33 conditions with different protocols and extraction methods. We detected no evidence of batch effects in replicate DNA samples or extractions from the same stool sample. Variability in DNA yield and alpha diversity was observed between the different collection, handling, and processing protocols. However, across all protocols, subject variability was the dominant contributor to microbiome structure, with comparatively little impact of the protocol used. While collection method and DNA extraction kit may affect DNA yield, and correspondingly alpha diversity, our findings suggest that characterization of the structure and composition of the fecal microbiome of infants and young children are reliably measurable by standardized collection, handling, and processing protocols and DNA extraction methods within an individual longitudinal study.

SUBMITTER: Antosca K

PROVIDER: S-EPMC7221451 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children.

Antosca Katherine K Hoen Anne G AG Palys Thomas T Hilliard Margaret M Morrison Hilary G HG Coker Modupe M Madan Juliette J Karagas Margaret R MR

MicrobiologyOpen 20200312 5

With the emergence of large-scale epidemiologic human microbiome studies, there is a need to understand the reproducibility of microbial DNA sequencing and the impact of specimen collection and processing methods on measures of microbial community composition and structure, with reproducibility studies in infants and young children particularly lacking. Here, we examined batch-to-batch variability and reliability of collection, handling, and processing protocols, testing replicate stool samples ...[more]

PMID: 32166902

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Project description:BackgroundOur aim was to evaluate infant behavioral state, stool microbiome profile and calprotectin in infants with infantile colic receiving a partially hydrolyzed protein formula with or without added Lacticaseibacillus (formerly Lactobacillus) rhamnosus GG (LGG).MethodsIn this single-center, double-blind, controlled, parallel, prospective study, term infants (14-28 days of age) identified with colic (using modified Wessel's criteria: cried and/or fussed ≥ 3 h/day for ≥ 3 days/week, in a one-week period) were randomized to receive one of two formulas over a three-week feeding period: marketed partially hydrolyzed cow's milk-based infant formula (PHF, n = 35) or a similar formula with added LGG (PHF-LGG, n = 36). Parent-reported infant behavior was recorded at three time points (Study Days 2-4, 10-12, and 18-20). Duration (hours/day) of crying/fussing (averaged over each three-day period) was the primary outcome. Stool samples were collected at Baseline and Study End (Days 19-21) to determine stool LGG colonization (by qPCR) and microbial abundance (using 16S rRNA gene sequencing) and calprotectin (μg/g).ResultsDuration of crying/fussing (mean ± SE) decreased and awake/content behavior increased over time with no significant group differences over the course of the study. There were no group differences in the percentage of infants who experienced colic by study end. Colic decreased by Study End vs Baseline in both groups. Change in fecal calprotectin also was similar between groups. Comparing Study End vs Baseline, LGG abundance was greater in the PHF-LGG group (P < 0.001) whereas alpha diversity was greater in the PHF group (P = 0.022). Beta diversity was significantly different between PHF and PHF-LGG at Study End (P = 0.05). By study end, relative abundance of L. rhamnosus was higher in the PHF-LGG vs PHF group and vs Baseline.ConclusionsIn this pilot study of infants with colic, both study formulas were well tolerated. Crying/fussing decreased and awake/content behavior increased in both study groups over the course of the study. Study results demonstrate a successful introduction of the probiotic to the microbiome. The partially hydrolyzed protein formula with added LGG was associated with significant changes in the gut microbiome.Trial registrationClinicalTrials.gov, ClinicalTrials.gov Identifier: NCT02340143 . Registered 16/01/2015.

Project description:Early childhood is a critical time for establishing food preferences and dietary habits. In order for appropriate advice to be available to parents and healthcare professionals it is essential for researchers to understand the ways in which children learn about foods. This review summarizes the literature relating to the role played by known developmental learning processes in the establishment of early eating behavior, food preferences and general knowledge about food, and identifies gaps in our knowledge that remain to be explored. A systematic literature search identified 48 papers exploring how young children learn about food from the start of complementary feeding to 36 months of age. The majority of the papers focus on evaluative components of children's learning about food, such as their food preferences, liking and acceptance. A smaller number of papers focus on other aspects of what and how children learn about food, such as a food's origins or appropriate eating contexts. The review identified papers relating to four developmental learning processes: (1) Familiarization to a food through repeated exposure to its taste, texture or appearance. This was found to be an effective technique for learning about foods, especially for children at the younger end of our age range. (2) Observational learning of food choice. Imitation of others' eating behavior was also found to play an important role in the first years of life. (3) Associative learning through flavor-nutrient and flavor-flavor learning (FFL). Although the subject of much investigation, conditioning techniques were not found to play a major role in shaping the food preferences of infants in the post-weaning and toddler periods. (4) Categorization of foods. The direct effects of the ability to categorize foods have been little studied in this age group. However, the literature suggests that what infants are willing to consume depends on their ability to recognize items on their plate as familiar exemplars of that food type.

Dataset Information

Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children.

Publications

Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children.

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