Estimation of donor-derived cell-free DNA ratio by SNP capture hybridization and clustering of NGS data

Measurement of donor-derived cell-free DNA (dd-cfDNA) enables early, non-invasive monitoring of transplanted organs, including rejection detection. We developed a method to estimate dd-cfDNA ratios by combining capture hybridization of 300 SNPs, next-generation sequencing (NGS), and clustering analysis. Validation was performed using simulated mixtures of fragmented genomic DNA from two individuals (0–100%). dd-cfDNA ratios were estimated via clustering, with and without 0% mixture samples, simulating the availability of pre-transplant recipient plasma. Inclusion of 0% samples yielded an r² of 0.9987 across the full 0–100% range; without them, r² remained high (0.9896) within the clinically relevant 0–10% range. We also compared clustering-based estimates with directly calculated values from kidney transplant pairs, in which donor and recipient SNP genotypes were known and could be inferred from read counts. The concordance correlation coefficient (CCC) for dd-cfDNA from day 0 to day 28 post-transplantation was 0.9887 and 0.9316 for unrelated pairs with and without pre-transplant data, respectively. For sibling pairs, CCCs were 0.9923 and 0.9675. For parent–child pairs, CCC was 0.9831 with pre-transplant data. CCC was not calculated without pre-transplant data due to limited data points (< 10%, n = 3). These results demonstrate high concordance and support the usefulness of our clustering-based method.