A comprehensive benchmarking study of protocols and sequencing platforms for 16S rRNA community profiling

Area of Science:

• Microbiology
• Bioinformatics
• Genomics

Background:

• Rapid advancements in sequencing technologies have transformed metagenomic and metagenetic studies.
• Benchmarking methodologies is essential for understanding microbial community analysis strengths and limitations.
• The 16S ribosomal RNA gene is a key phylogenetic marker, with studies evolving to interrogate thousands of samples and multiple gene regions.

Purpose of the Study:

• To benchmark various 16S rRNA gene sequencing methodologies and experimental designs.
• To assess the performance of leading sequencing platforms (Illumina MiSeq, PacBio RSII, Roche 454, IonTorrent).
• To evaluate the impact of library preparation and primer choice on microbial community profiling.

Main Methods:

• Utilized two synthetic microbial communities (even and uneven distribution) as control material.
• Tested three experimental designs (fusion primers, universal tailed tag, ligated adaptors) across nine hypervariable 16S rDNA regions.
• Analyzed data from multiple next-generation sequencing platforms.

Main Results:

• Library preparation significantly impacts data interpretation due to varying error and chimera rates.
• Observed community composition showed platform- and primer-dependent biases.
• Despite biases, 16S rRNA sequencing remains quantitative for recovering relative taxa abundance changes.

Conclusions:

• Choice of sequencing platform and experimental design requires early-stage consideration and pilot testing.
• Using synthetic communities as positive controls aids in identifying potential biases and procedural drawbacks.
• This study provides guidelines for selecting optimal experimental conditions and sequencing platforms for microbial profiling.