Reproducibility and repeatability of six high-throughput 16S rDNA sequencing protocols for microbiota profiling

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Culture-independent molecular techniques and next-generation sequencing (NGS) enable large-scale microbiota studies.
• Achieving high reproducibility and repeatability in NGS is crucial for reliable epidemiological data.

Purpose of the Study:

• To assess the reproducibility of saliva microbiota profiling using triplicate samples.
• To compare different 16S amplicon assay variations for optimal microbiota analysis.

Main Methods:

• In-house 16S amplicon assays were developed with TruSeq (TS-tailed) or Nextera (NX-tailed) adapters.
• Assays included dual indexing, with or without a 6-nt internal index.
• Triplicate saliva samples were analyzed to evaluate reproducibility across protocols.

Main Results:

• All amplification protocols yielded consistent microbial profiles for identical samples.
• The TS-tailed method demonstrated the highest reproducibility in this study.
• The TS-tailed 1-step protocol on the HiSeq platform provided high alpha-diversity and low variability.

Conclusions:

• Large-scale microbiota profiling is consistently achievable with various 16S amplicon assays.
• The TS-tailed-1S dual index protocol is recommended for its repeatable results and reduced labor intensity on the HiSeq platform.