Improved next-generation sequencing pre-capture library yields and sequencing parameters using on-bead PCR.

Tumor DNA sequencing results can have important clinical implications. However, its use is often limited by low DNA input, owing to small tumor biopsy size. To help overcome this limitation we have developed a simple improvement to a commonly used next-generation sequencing (NGS) capture-based library preparation method using formalin-fixed paraffin-embedded-derived tumor DNA. By using on-bead PCR for pre-capture library generation we show that library yields are dramatically increased, resulting in decreased sample failure rates. Improved yields allowed for a reduction in PCR cycles, which translated into improved sequencing parameters without affecting variant calling. This methodology should be applicable to any NGS system in which input DNA is a limiting factor. Lay abstract The results of tumor DNA sequencing can have important clinical implications. However, limited DNA input owing to small tumor size frequently results in a failed analysis. By incorporating a simple modification to the standard sample preparation method, we show that DNA yields can be greatly increased, allowing for more samples to be successfully sequenced. Further, the methodology used also results in a subsequent improvement in sequencing parameters, with no loss in the ability to detect mutations. METHOD SUMMARY To improve the library yield for low DNA input tumor samples subjected to NGS, we adopted a simple modification of the standard KAPA Hyper library preparation procedure. Genomic DNA libraries were subjected to a solid-phase reversible mobilization bead-based cleanup; however, they were not eluted from the beads prior to pre-capture PCR. Post-PCR cleanup was performed using a 20% PEG, 2.5M NaCl buffer, resulting in a dramatic yield increase with no loss of sequencing fidelity.