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.

The effect of low-abundance OTU filtering methods on the reliability and variability of microbial composition assessed by 16S rRNA amplicon sequencing

“…Currently, there is no consensus regarding best practices for human stool collection and analysis, leading to concerns regarding the reliability and reproducibility of these datasets. Several factors including DNA extraction methods, sequencing technologies, and analytical approaches are known to have a significant impact on the characterization of the stool microbiota ( Lauber et al., 2010 ; Carroll et al., 2012 ; Goodrich et al., 2014 ; Sinha et al., 2015 ; Chiu and Chao, 2016 ; Sinha et al., 2017 ; Vogtmann et al., 2017 ; Antosca et al., 2020 ; Bartolomaeus et al., 2020 ), underscoring the importance of using standardized protocols to minimize bias.…”

The acidified drinking water-induced changes in the behavior and gut microbiota of wild-type mice depend on the acidification mode

“… 31 and Antosca et al . 32 . We also found that mouse fecal samples collected, sequenced and analyzed 3 years apart had very similar microbiota compositions (Fig.…”

“…Sequencing platforms including the Illumina MiSeq we used has sequencing errors, and separate sequencing runs may contribute to differences in the gut microbiota compositions. However, the reproducibility of Illumina MiSeq sequencing for microbiota analysis have been evaluated and high reproducibility was shown by Caporaso et al 29 , Eriksson et al 30 , Teng et al 31 and Antosca et al 32 . We also found that mouse fecal samples collected, sequenced and analyzed 3 years apart had very similar microbiota compositions (Fig.…”

Prospective association of the infant gut microbiome with social behaviors in the ECHO consortium

“…Furthermore, SRS-2 scores have high consistency over time, minimizing the effect of age at which the surveys were administered in the two cohorts [ 57 ]. Another limitation is the variation in stool sample collection methods between the two studies, including the timing of sample collection, which may have led to differential detection of certain species; much of the concern about collection and processing methods may be alleviated with a rigorous comparison study of methods identifying data alignment with variable methods [ 58 ].…”