Comparative Analysis of 16S rRNA Gene and Metagenome Sequencing in Pediatric Gut Microbiomes

Abstract

The colonization of the human gut microbiome begins at birth, and over time, these microbial communities become increasingly complex. Most of what we currently know about the human microbiome, especially in early stages of development, was described using culture-independent sequencing methods that allow us to identify the taxonomic composition of microbial communities using genomic techniques, such as amplicon or shotgun metagenomic sequencing. Each method has distinct tradeoffs, but there has not been a direct comparison of the utility of these methods in stool samples from very young children, which have different features than those of adults. We compared the effects of profiling the human infant gut microbiome with 16S rRNA amplicon vs. shotgun metagenomic sequencing techniques in 338 fecal samples; younger than 15, 15–30, and older than 30 months of age. We demonstrate that observed changes in alpha-diversity and beta-diversity with age occur to similar extents using both profiling methods. We also show that 16S rRNA profiling identified a larger number of genera and we find several genera that are missed or underrepresented by each profiling method. We present the link between alpha diversity and shotgun metagenomic sequencing depth for children of different ages. These findings provide a guide for selecting an appropriate method and sequencing depth for the three studied age groups.

Undervalued Pseudo- nifH Sequences in Public Databases Distort Metagenomic Insights into Biological Nitrogen Fixers

“…Additionally, we assessed how pseudo- nifH sequences in public databases affect metagenomic analyses of environmental samples. We focused on reusable metagenomic data sets in NCBI SRA/EMBL-EBI ERA/DDBJ DRA ( 51 ) under the following environmental categories: “activated sludge metagenome,” “human gut metagenome,” “termite metagenome,” “wastewater metagenome,” and “∗ sediment metagenome” ( 52 – 64 , 72 ). We randomly picked SRA/ERA/DRA accession numbers that satisfy the following criteria: (i) sequenced on Illumina MiSeq, HiSeq, MiniSeq, NextSeq, or NovaSeq (i.e., not subject to frameshifting read errors by Roche 454); (ii) labeled as a “WGS” (standing for whole-genome shotgun) project; and (iii) described in a peer-reviewed literature (i.e., likely to be technically sound).…”

Reconstitution and Transmission of Gut Microbiomes and Their Genes between Generations

“…Although these are powerful and valuable tools, they have limitations. For example, profiling gut microbiomes by both 16S rRNA gene sequencing and shotgun metagenomic sequencing techniques demonstrated several genera that are missed or underrepresented by each profiling method separately [270].…”

Microbiome.jl and BiobakeryUtils.jl - Julia packages for working with microbial community data

“…While Microbiome.jl and BiobakeryUtils.jl are already functional and being used for research (Lewis et al, 2021;Peterson et al, 2021;Tso et al, 2021), there are several avenues for further development.…”