This study investigates the sources of bias in Illumina sequencing libraries, particularly focusing on the PCR amplification step. The authors used quantitative PCR (qPCR) to trace a diverse panel of loci through the Illumina library preparation process, identifying PCR as the primary source of base-composition bias. They optimized PCR conditions, including extending the denaturation step and using AccuPrime Taq HiFi polymerase, to reduce bias and improve the representation of GC-rich and AT-rich loci. The optimized protocol significantly reduced amplification bias and minimized the effects of PCR instrument and temperature ramp rate. The study also compared PCR-amplified and PCR-free libraries, finding that PCR-free libraries performed equally well but required more input DNA and were less practical for many sample types. The optimized PCR conditions are easy to implement in high-throughput production and do not increase DNA input requirements, making them suitable for routine Illumina library construction.This study investigates the sources of bias in Illumina sequencing libraries, particularly focusing on the PCR amplification step. The authors used quantitative PCR (qPCR) to trace a diverse panel of loci through the Illumina library preparation process, identifying PCR as the primary source of base-composition bias. They optimized PCR conditions, including extending the denaturation step and using AccuPrime Taq HiFi polymerase, to reduce bias and improve the representation of GC-rich and AT-rich loci. The optimized protocol significantly reduced amplification bias and minimized the effects of PCR instrument and temperature ramp rate. The study also compared PCR-amplified and PCR-free libraries, finding that PCR-free libraries performed equally well but required more input DNA and were less practical for many sample types. The optimized PCR conditions are easy to implement in high-throughput production and do not increase DNA input requirements, making them suitable for routine Illumina library construction.