PCR bias impacts microbiome ecological analyses.

Polymerase Chain Reaction (PCR) is a critical step in amplicon-based microbial community profiling, allowing the selective amplification of marker genes such as 16S rRNA from environmental or host-associated samples. Despite its widespread use, PCR is known to introduce amplification bias, where some DNA sequences are preferentially amplified over others due to factors such as primer-template mismatches, sequence GC content, and secondary structures. Although these biases are known to affect transcript abundance, their implications for ecological metrics remain poorly understood. In this study, we conduct a comprehensive evaluation of how PCR-bias influences both within-samples (α-diversity) and between-sample (β-diversity) analyses. We show that perturbation-invariant diversity measures remain unaffected by PCR bias, but widely used metrics such as Shannon diversity and Weighted-Unifrac are sensitive. To address this, we provide theoretical and empirical insight into how PCR-induced bias varies across ecological analyses and community structures, and we offer practical guidance on when bias-correction methods should be applied. Our findings highlight the importance of selecting appropriate diversity metrics for PCR-based microbial ecology workflows and offer guidance for improving the reliability of diversity analyses.