illumina/DNA PCR-free WGS

This protocol describes PCR-free whole genome sequencing library preparation optimized for Illumina NovaSeq 6000 and NovaSeq X platforms. By eliminating the PCR amplification step, these libraries provide more uniform coverage and reduced GC-bias compared to traditional PCR-based approaches.

Overview

The workflow involves mechanical DNA fragmentation using a Covaris ultrasonicator, end repair and A-tailing, adapter ligation with unique dual indexes, and double-sided size selection. The resulting libraries have a target insert size of 350 bp and are ready for cluster generation without amplification.

Key considerations

• Input DNA: Requires 100–500 ng of high-quality, high-molecular-weight genomic DNA (DIN > 7.0)
• Fragment size: Target 350 bp insert size for optimal paired-end sequencing on NovaSeq
• Coverage: Recommend 30x for human whole genome; adjust loading concentration accordingly
• Quality control: Final libraries should show a single peak at ~450 bp (insert + adapters) on Bioanalyzer