A scalable and cost-effective in-line barcoding strategy for standardized 16S rRNA gene amplicon sequencing: performance evaluation and bias assessment

In-line barcoding offers a streamlined and scalable alternative to two-step PCR library preparation for 16S rRNA gene amplicon sequencing, enabling cost-effective, high-throughput profiling of microbial communities. Here, we tested 136 and 156 in-line barcoded primer pairs for bacterial and archaeal communities for their performance across three environmental samples and a mock standard community using both PCR-free and PCR-based adapter ligation workflows and with different pooling strategies. The primers were designed by combining widely used universal 16S rRNA gene primers with existing barcode sets from Illumina kits. The designed primer pairs produced efficient and consistent amplification with minimal dropout and no systematic taxonomic bias. While minor variation in read distribution was observed, overall community structure and diversity metrics remained robust to barcode identity. The PCR-free adapter ligation protocol demonstrated superior read recovery and reproducibility, especially for low-diversity archaeal samples, compared to the PCR-based protocol. Through clustering and performance-based filtering, we selected final sets of 96 pairs for both bacterial and archaeal communities that work efficiently and well together for direct further use. This in-line tagging strategy is easy to adopt with fewer processing steps and PCR-associated artifacts, allows straightforward sample tracking, and supports reliable large-scale microbiome studies. We also present a framework for evaluating barcode- or primer-induced biases. All validation datasets, open-source processing scripts, and barcode design resources are provided to promote reproducibility and community-wide adoption.