Impact of Primer and Template Designs on PCR and NGS Performance in Single‐Stranded DNA‐Encoded Library Screening at Different Inputs

DNA‐Encoded Library (DEL) screenings require sufficient copies per library member to ensure reliable hit identification and reproducibility across experiments. Here, we screen various primers and template DNA sequences for PCR amplification and next‐generation sequencing (NGS), aiming to lower the required library input while maintaining robust hit discovery rates. Analysis of selection fingerprints using an optimized primer design shows that the minimum input required to identify hits can be reduced to 10 3 copies per member, representing a 100‐fold decrease relative to prior reports. This optimization is particularly impactful for large libraries containing billions of compounds, where increased synthesis costs and larger absolute quantities required for selections necessitate minimizing the amount consumed per selection experiment.