A protocol for characterization of extremely preterm infant gut microbiota in double-blind clinical trials

Summary

16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials.

For complete details on the use and execution of this protocol, please refer to Martí et al. (2021) and Spreckels et al. (2021).

Graphical abstract

Highlights

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  Customizable protocol for characterization of infant gut microbiota in clinical trials

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  Optimized sample preparation for 16S rRNA gene sequencing for infant fecal samples

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  Quantitative PCR for probiotic Lactobacillus reuteri quantification in infant feces

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  Easy-to-use bioinformatic and statistical analysis pipeline

Abstract

16S rRNA gene sequencing enables microbial community profiling, but recovering fecal DNA from extremely premature infants is challenging. Here, we describe an optimized protocol for fecal DNA isolation, library preparation for 16S rRNA gene sequencing, taxonomy assignation, and statistical analyses. The protocol is complemented with a quantitative PCR for probiotic L. reuteri identification. This protocol describes how to characterize preterm infant gut microbiota and relate it to probiotic supplementation and clinical outcomes. It is customizable for other clinical trials.