Protocol optimization of a targeted sequencing panel for genomic profiling of bronchoalveolar lavage fluid in lung cancer

IntroductionWe investigated a commercially available sequencing panel to study the effect of sequencing depth, variant calling strategy, and targeted sequencing region on identifying tumor-derived variants in cell-free bronchoalveolar lavage (cfBAL) DNA compared with plasma cfDNA.MethodsSequencing was performed at low or high coverage using two filtering algorithms to identify tumor variants on two panels targeting 77 and 197 genes respectively.ResultsOne hundred and four sequencing files from 40 matched DNA samples of cfBAL, plasma, germline leukocytes, and archival tumor specimens in 10 patients with early-stage lung cancer were analyzed. By low-coverage sequencing, tumor-derived cfBAL variants were detected in 5/10 patients (50%) compared with 2/10 (20%) for plasma. High-coverage sequencing did not affect the number of tumor-derived variants detected in either biospecimen type. Accounting for germline mutations eliminated false-positive plasma calls regardless of coverage (0/10 patients with tumor-derived variants identified) and increased the number of cfBAL calls (5/10 patients with tumor-derived variants identified). These results were not affected by the number of targeted genes.