Large scale differences in characterization of gut microbiome between 16S amplicon and shallow shotgun sequencing

Abstract

Introduction: The advent of next generation sequencing (NGS) technology aided exponentially to the understanding of the gut microbiome. The most commonly used approaches of NGS are 16S rRNA and shotgun metagenome sequencing. 16S rRNA sequencing provides microbial resolution only up to the genus level, while deep shotgun metagenome sequencing provides taxonomic profiling down to the species level but the high cost hampers its usage. Alternate method, Shallow shotgun metagenome sequencing (SSMS) emerged as a cost effective high throughput method for microbial profiling.

Objective: To compare the consistency of 16S rRNA and SSMS NGS methods in gut microbial profiling.

Materials & Methods: DNA was extracted from the stool samples of five infants, and the full 16S rRNA region was amplified. Sequencing libraries were prepared for 16S rRNA sequencing and SSMS, loaded onto the flow cell, sequenced using the MiSeq platform (Illumina), and analyzed through custom bioinformatics analysis pipeline.

Results: NGS data analysis revealed that gut microbiome characterized using both 16S rRNA sequencing and SSMS showed large scale differences in terms of reported species. Some of the microbes detected using 16S rRNA sequencing were found to be missing while using SSMS and vice versa. Very few microbes were commonly detected by both sequencing approaches.

Conclusion: The application of both NGS sequencing approaches is warranted for accurate bacterial profiling from different sources.

Keywords: Gut Microbiome; 16S rRNA Sequencing; Shallow Shotgun Metagenomic Sequencing; Microbial Profiling.