Aim: Monitoring the health of an organ following transplantation is crucial to the selection of timely and optimal treatment strategies. The One Lambda™ Devyser Accept cfDNA kit enables detection of cell-free DNA (cfDNA) from up to 3 genotypes in circulation following kidney transplantation. To accurately measure the genotype-of-origin proportions of circulating cfDNA, cfDNA isolated from plasma must be of sufficient purity and concentration. The objective of this study was to assess the compatibility of different cfDNA extraction method with the One Lambda™ Devyser Accept cfDNA kit.
Method: Three cfDNA extraction kits- EZ1&2 ccfDNA Kit (Qiagen), MagMAX Cell-Free DNA Kit (Thermo Fisher), and Maxwell RSC ccfDNA LV Plasma Kit (Promega), automated on corresponding instruments - were utilized. Each extraction platform was evaluated using three plasma samples from 10 donors, and artificial plasma (Biochemazone) spiked with sheared genomic DNA and interfering substances. Quality and yield of cfDNA were assessed, and libraries were generated for downstream sequencing. Variant allele frequency (VAF) compliance and substance interference were analyzed.
Results: cfDNA samples obtained with the Qiagen EZ1 kit passed extraction and sequencing quality control, with occasional bead carry-over requiring removal. cfDNA purified with Thermo Fisher MagMAX / KingFisher Flex passed criteria but required elution in maximum volume for consistent top quality library generation. cfDNA obtained with Promega Maxwell passed criteria with proteinase K pre-treatment implemented, and a 1:1 dilution with elution buffer improved PCR amplification. All kits produced cfDNA suitable for downstream NGS library generation and accurate measurement of cfDNA proportions.
Conclusion: The study demonstrates that the Qiagen EZ1 kit requires no modifications, while slight adjustments are needed for MagMAX and Maxwell kits to produce consistent high-quality cfDNA for downstream NGS analysis. These findings support the use of different extraction method with the One Lambda Devyser Accept cfDNA assay.
