Smart non-uniformity: Calibration of sequencing depth of a targeted gene panel to simultaneously detect somatic and germline variants.

Targeted gene panel sequencing that measures genomic variation at different depths has potential diagnostic application. A targeted gene panel was developed to detect 1) somatic variants associated with clonal haematopoiesis of indeterminate potential (CHIP), which requires an optimal sequencing depth of >500X, and 2) germline variants requiring a lower ≥50X depth [Panel 1]. This was achieved by adjusting probe ratios for genomic regions relevant to identifying CHIP in comparison to those relevant to germline variation analysis. An additional custom panel containing only the genomic regions relevant to the identification of CHIP [Panel 2] was also manufactured to confirm that panel 1 did not miss variants due to the complex design. Both panels were used to sequence 150 blood-derived DNAs; 94 DNAs from research participants aged 64-75 years; 16 DNAs with known germline variants; 16 DNAs with known germline variants (titrated from 0-100%); 24 DNAs from individuals aged <40 years and three commercial CHIP controls and three high molecular weight DNA controls. The sequencing median depth ratio between the CHIP and germline relevant genomic regions was 4.7:1. Fourteen CHIP-associated variants were called in both Panel 1 (1,382X median variant-depth) and Panel 2 (1,665X median variant-depth). All known germline variants were identified (251X median variant-depth). Smart non-uniformity sequencing reliably detects variants with allele frequency in the range >0.01-1 in one workflow.