Inconsistent Patterns of Microbial Diversity and Composition Between Highly Similar Sequencing Protocols: A Case...

Area of Science:

• Microbiology
• Bioinformatics
• Ecology

Background:

• 16S rRNA gene sequencing is widely used for microbial community analysis.
• Standardized protocols are assumed to produce comparable results, enabling meta-analyses.
• Previous studies have not fully explored protocol-specific variations in microbiome data.

Purpose of the Study:

• To evaluate the consistency of microbiome data generated by slightly different 16S rRNA gene sequencing protocols.
• To challenge the assumption that data from varying protocols are directly comparable for microbiome analysis.
• To investigate the impact of protocol differences on diversity metrics in coral-associated microbial communities.

Main Methods:

• Technical replicates of coral samples (Montipora aequituberculata and Porites lobata) were used.
• Two distinct 16S rRNA gene sequencing protocols differing in DNA polymerase and sequencing platform were applied.
• Alpha and beta diversity metrics were calculated and compared between the datasets.
• Analyses were repeated after removing low-abundance taxa and at higher taxonomic levels.

Main Results:

• Minimal variation in alpha diversity was observed between protocols.
• Significant differences in beta diversity metrics were detected, dependent on host species.
• These inconsistencies persisted across different data filtering and taxonomic aggregation levels.
• Protocol-dependent bacterial community differences were context-specific and difficult to correct.

Conclusions:

• Combining 16S rRNA gene sequence data from distinct protocols requires caution.
• Protocol variations can introduce significant biases, particularly in beta diversity.
• Further research is needed to understand the mechanistic causes of these observed differences.
• Validation is crucial before integrating microbiome data from disparate protocols.