Estimating mutation load at 5% LOD from FFPE samples using a targeted next-generation sequencing assay.

28Background: High tumor mutation load is a biomarker which positively correlates with response to immune checkpoint inhibitors. Current methods to estimate tumor mutation load often require large amounts of DNA. Herein, we demonstrate the ability of a targeted panel to estimate mutation load from FFPE samples using low input amount of DNA. Methods: We developed a single-sample analysis workflow to estimate mutation load (somatic mutation count per Megabase (Mb)) from FFPE and fresh frozen tumor research samples. The assay utilizes a PCR-based target enrichment panel that covers ~1.7 Mb. Our workflow requires only 10 ng of input DNA, and enables a 2.5-day turn-around time from sample to the final report. The workflow enables < 60 minutes of hands-on time for automated library preparation and templating on a batch of 8 samples. Sequencing is performed using a high throughput semiconductor sequencing platform to achieve sufficient depth (~500x coverage) and accuracy. Our analysis pipeline calls variants wit...