Variations in 16S rRNA-based microbiome profiling between pyrosequencing runs and between pyrosequencing facilities.

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Pyrosequencing of 16S rRNA gene amplicons is a common method for microbiome analysis.
• Variations in sequencing runs or facilities can lead to differing microbiome profiles.

Purpose of the Study:

• To evaluate variations in microbiome profiling between different pyrosequencing runs and facilities.
• To identify sources of variability in 16S rRNA gene amplicon sequencing.

Main Methods:

• Sequencing of 16S rRNA gene amplicon libraries from rumen samples.
• Utilizing the 454 FLX Titanium platform at two independent sequencing facilities.
• Replicating sequencing runs for each sample set.

Main Results:

• Similar relative abundances were observed for predominant taxa but not for minor taxa.
• Distinct bacterial profiles were found between the two sequencing facilities, affecting both predominant and minor taxa.
• Variations were noted in the abundance of Prevotella, Lachnospiraceae, and Proteobacteria.

Conclusions:

• Differences in sequencing runs and facilities significantly impact microbiome profiling results.
• Primer choice and 16S rRNA gene regions used are likely major contributors to observed variations.
• Quantitative interpretation of relative taxon abundance, particularly for minor taxa, requires caution, and cross-study comparisons should be made carefully.