A Scalable and Cost-Effective In-Line Barcoding Strategy for Standardized 16S rRNA Gene Amplicon Sequencing:...

Area of Science:

• Microbiology
• Genomics
• Bioinformatics

Background:

• Traditional two-step PCR library preparation for 16S rRNA gene amplicon sequencing is complex and costly.
• High-throughput microbial community profiling requires streamlined and scalable methods.

Purpose of the Study:

• To develop and validate in-line barcoded primer pairs for efficient bacterial and archaeal 16S rRNA gene amplicon sequencing.
• To establish a framework for evaluating primer- and barcode-induced biases in amplicon sequencing.

Main Methods:

• Designed and tested 136 bacterial and 156 archaeal in-line barcoded primer pairs by combining universal 16S rRNA primers with existing Illumina barcodes.
• Evaluated primer pair performance on environmental samples and a mock community.
• Selected optimal primer sets through clustering and performance-based filtering.

Main Results:

• Developed efficient and consistent primer pairs with minimal dropout and no systematic taxonomic bias.
• Selected 96 optimized pairs for bacterial and 96 for archaeal communities.
• Demonstrated the in-line barcoding strategy's ease of adoption, reduced PCR artifacts, and improved sample tracking.

Conclusions:

• In-line barcoding provides a cost-effective, scalable, and reliable method for high-throughput microbial community analysis.
• The strategy is adaptable to various amplicon sequencing applications beyond 16S rRNA surveys.
• Provided resources promote reproducibility and community adoption of the in-line barcoding approach.