International interlaboratory study comparing single organism 16S rRNA gene sequencing data: Beyond consensus...

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• 16S ribosomal RNA (rRNA) gene sequencing is crucial for bacterial identification.
• Interlaboratory variability in sequencing data can hinder accurate microbial analysis.
• Multi-copy, paralogous genes present unique challenges for sequencing data comparison.

Purpose of the Study:

• To develop and evaluate a high-resolution method for comparing sequencing data from different platforms for multi-copy genes.
• To assess interlaboratory reproducibility of 16S rRNA gene sequencing for bacterial identification.
• To establish analytical methods for evaluating variant combinations in gene families.

Main Methods:

• Six laboratories performed 16S rRNA gene sequencing on Escherichia coli and Listeria monocytogenes strains using Sanger, Roche 454, and Ion Torrent PGM platforms.
• Sequencing data were evaluated for conserved positions, variant copy ratios, and variant combinations.
• Bayesian and maximum likelihood statistics were employed to estimate variant ratios and combinations.

Main Results:

• Conserved positions in the 16S rRNA gene were consistently identified across all sequencing methods.
• Reproducibility of variant copy ratios was achieved only with high-throughput sequencing methods.
• Accurate identification of variant combinations required increased sequencing depth and longer read lengths.

Conclusions:

• High-throughput sequencing technologies are essential for reproducible analysis of multi-copy genes.
• Sequencing depth and read length are critical factors for resolving genetic variants in complex gene families.
• Novel analytical approaches improve the evaluation of multi-copy gene sequence data across diverse platforms and laboratories.