Metagenomic Information Recovery from Human Stool Samples Is Influenced by Sequencing Depth and Profiling Method

Area of Science:

• Microbiome research
• Metagenomics
• Bioinformatics

Background:

• 16S rRNA gene sequencing is a common but limited method for microbiome analysis.
• Shotgun metagenomic sequencing (SMS) provides deeper insights but is more costly.
• Shallow shotgun metagenomic sequencing (SSMS) offers a cost-effective alternative with improved resolution.

Purpose of the Study:

• To evaluate the impact of sequencing depth on SSMS for human stool microbiome analysis.
• To compare marker gene-mapping and alignment-based annotation methods.
• To assess the recovery of bacterial taxa, viruses, and functional pathways at varying depths.

Main Methods:

• Analysis of healthy human stool samples using SSMS at different sequencing depths.
• Application of marker gene-mapping and alignment-based annotation tools.
• Quantification of identified taxa, viruses, and metabolic pathways.

Main Results:

• Taxa identification decreased significantly with reduced sequencing depth, especially for marker gene-mapping.
• Viral and pathway recovery demonstrated a clear dependence on sequencing depth.
• Annotation accuracy and completeness are influenced by both sequencing depth and chosen methodology.

Conclusions:

• SSMS provides valuable species-level and functional data for microbiome studies.
• Sequencing depth is a critical factor influencing the reliability of SSMS results.
• Understanding these depth-dependent effects is crucial for accurate metagenomic analysis and tool selection.