Evaluation of pre-analytical factors affecting plasma DNA analysis

Circulating tumor DNA analysis has several potential applications in cancer diagnostics. However, results in literature vary considerably, often due to different blood collection methods and protocols. Several new products to address pre-analytical variables of cfDNA processing have recently become available, and little is understood about their effects on DNA quality and downstream applications. We evaluated the effects of blood collection protocols and DNA extraction kits on cfDNA yield, quality, and fragment size using droplet-digital PCR (ddPCR) and targeted deep sequencing. To quantify cfDNA yield and size distribution, we developed a multiplexed ddPCR assay with FAM-labeled short amplicons and TET-labeled long amplicons targeting housekeeping genes. After assay validation, we compared the performance of several commercially-available cfDNA extraction kits using control plasma samples and different blood collection protocols using paired plasma samples from healthy volunteers. To assess whether cell-stabilizing preservative in Streck tubes may induce low-abundance noise in cfDNA, we performed molecularly-tagged targeted deep sequencing and developed an informatics approach for enumeration and variant calling from uniquely tagged DNA fragments. We found a significant difference between extraction methods but no significant difference across blood collection protocols in cfDNA yield or size distribution. Sequencing results showed no significant evidence of preservative-induced cfDNA damage across tested blood collection protocols. In summary, the multiplexed ddPCR assay enabled quantitative assessment of cfDNA extraction methods and blood collection protocols, and allowed for normalization of input to create reproducible sequencing libraries. Our results suggest that plasma samples processed up to 72 hours following venipuncture in Streck Cell-free DNA tubes may be used for downstream sequencing of circulating tumor DNA in patients with cancer.