Reliability of stool microbiome methods for DNA yields and sequencing among infants and young children. Issue 5 (12th March 2020)

• Record Type: Journal Article
• Authors: Antosca, Katherine; Hoen, Anne G.; Palys, Thomas; Hilliard, Margaret; Morrison, Hilary G.; Coker, Modupe; Madan, Juliette; Karagas, Margaret R.
• Is Part Of: MicrobiologyOpen. Volume 9:Issue 5(2020)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2020-03-12
• Subjects: cohort studies -- gut microbiome -- infants -- reproducibility; Microbiology -- Periodicals; 579
• DOI: 10.1002/mbo3.1018
• Languages: English
• ISSNs: 2045-8827

Abstract

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. Abstract : Study question : What is the intralaboratory reliability of microbiome sequencing of infant stool samples and how do results differ with the various collection, handling, and processing extraction protocols? What is already known : Various protocols are used in human microbiome research. Studies of the reliability of these methods have been conducted largely among adults in whom the microbiome is relatively stable. What this study adds : Large‐scaled epidemiologic studies of the human microbiome are beginning to emerge. This study adds to evidence of the reliability of methods used to assess the early developing microbiome during the time of the greatest interperson variability.