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.
Highlights
There is increasing evidence that changes in activity and diversity of the gut microorganisms are associated with the development of diseases and conditions such as type II diabetes (Hartstra et al, 2015; Lambeth et al, 2015), cancer (Marchesi et al, 2011; Bultman, 2014), and even depression (Foster and McVey Neufeld, 2013)
Amplified sequences are computationally binned and identified typically using either operational taxonomic unit (OTU) clustering, a method which clusters sequences based on percent sequence similarity, and more recently, methods such as amplicon sequence variant (ASV) identification (Callahan et al, 2016), or sub-operational taxonomic unit (Amir et al, 2017), methods which identify unique sequences and remove low quality reads and sequence artifacts, by using a probabilistic model to assess the probability that a rare sequence is a true biological variant
Alpha Diversity Increases With Age in Both 16S rRNA Gene- and Metagenomic-Profiled Samples
Summary
There is increasing evidence that changes in activity and diversity of the gut microorganisms are associated with the development of diseases and conditions such as type II diabetes (Hartstra et al, 2015; Lambeth et al, 2015), cancer (Marchesi et al, 2011; Bultman, 2014), and even depression (Foster and McVey Neufeld, 2013). The two most widely used culture-independent methods are amplicon sequencing, a method that amplifies variable regions of a highly conserved bacterial gene such as the 16S rRNA gene, and shotgun metagenomic sequencing, an approach that sequences all of the DNA present in a sample. While each of these methods have unique advantages, the tradeoffs of these methods in young children in the context of sequencing depth and coverage remain largely unexplored. Many taxa often cannot be resolved because the V4–V5 doesn’t provide enough nucleotide variability to resolve different taxa
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