Smart Nonuniformity for Calibrating 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, smart nonuniformity sequencing, was developed to detect somatic variants associated with clonal hematopoiesis of indeterminate potential (CHIP), which requires an optimal sequencing depth of >500×; and germline variants requiring a lower ≥50× 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 because of the complex design. Both panels were used to sequence 150 blood-derived DNAs; 94 DNAs from research participants aged 64 to 75 years; 16 DNAs with known germline variants; 16 DNAs with known germline variants (titrated from 0% to 100%); 24 DNAs from individuals aged <40 years; and 3 commercial CHIP controls and 3 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 (1382× median variant depth) and panel 2 (1665× median variant depth). All known germline variants were identified (251× median variant depth). Smart nonuniformity sequencing reliably detects variants with allele frequency in the range >0.01 to 1 in one workflow.