Quantitative evaluation of microbiome sequencing resolution under varying experimental conditions using defined mock...

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Accurate species-level resolution is vital for microbiome analysis.
• Comparing sequencing technologies requires systematic evaluation of their performance.
• Standardized methods are needed to assess the impact of experimental conditions on resolution.

Purpose of the Study:

• To quantitatively evaluate and compare the species-level resolution of different sequencing strategies (16S V3-V4, full-length 16S rRNA, and whole metagenome shotgun sequencing).
• To assess the influence of input DNA concentration and sequencing output on resolution.
• To provide insights for optimizing microbiome sequencing and data interpretation.

Main Methods:

• Utilized commercial and custom whole-cell mock communities (MCs) for evaluation.
• Tested whole metagenome shotgun sequencing (WMS) with 12 combinations of input DNA and output levels.
• Assessed taxonomic resolution using an adjusted F1-score, integrating sensitivity and abundance reproducibility.

Main Results:

• Whole metagenome shotgun sequencing (WMS) achieved over 90% true positive abundance ratio, significantly outperforming full-length 16S rRNA (16F, ~60%) and 16S V3-V4 (16P, <10%).
• Optimal species-level resolution was achieved with 10 ng input DNA and 10 gigabases output.
• Performance varied by taxon and condition; some species required high input/output, while others showed sensitivity to input levels.

Conclusions:

• Whole metagenome shotgun sequencing (WMS) provides superior species-level resolution for microbiome analysis.
• Sequencing configuration (input DNA, output) and taxon-specific characteristics critically impact detection performance.
• Results offer guidance for selecting appropriate sequencing strategies and interpreting microbiome data effectively.