Abstract
Pre-analytical factors can significantly affect circulating cell-free DNA (cfDNA) analysis. However, there are few robust methods to rapidly assess sample quality and the impact of pre-analytical processing. To address this gap and to evaluate effects of DNA extraction methods and blood collection tubes on cfDNA yield and fragment size, we developed a multiplexed droplet digital PCR (ddPCR) assay with 5 short and 4 long amplicons targeting single copy genomic loci. Using this assay, we compared 7 cfDNA extraction kits and found cfDNA yield and fragment size vary significantly. We also compared 3 blood collection protocols using plasma samples from 23 healthy volunteers (EDTA tubes processed within 1 hour and Cell-free DNA Blood Collection Tubes processed within 24 and 72 hours) and found no significant differences in cfDNA yield, fragment size and background noise between these protocols. In 219 clinical samples, cfDNA fragments were shorter in plasma samples processed immediately after venipuncture compared to archived samples, suggesting contribution of background DNA by lysed peripheral blood cells. In summary, we have described a multiplexed ddPCR assay to assess quality of cfDNA samples prior to downstream molecular analyses and we have evaluated potential sources of pre-analytical variation in cfDNA studies.
Highlights
Analysis of circulating cell-free DNA from plasma has several potential diagnostic applications in prenatal, transplant and cancer medicine[1,2,3,4]
We included 5 short PCR amplicons with mean product size of 71 bp and corresponding probes labeled with FAM as well as 4 long PCR amplicons with mean product size of 471 bp and corresponding probes labeled with TET (Fig. 1 and Supplemental Table 1)
We have developed a multiplexed droplet digital PCR approach to reliably assess amplifiable quantities of cell-free DNA (cfDNA) and estimate the contribution of high molecular weight background DNA in a single step
Summary
Analysis of circulating cell-free DNA from plasma (cfDNA) has several potential diagnostic applications in prenatal, transplant and cancer medicine[1,2,3,4]. High fractions of HMW DNA in plasma can complicate PCR and tagmentation-based sequencing because these methods will incorporate intact DNA in a sample, potentially biasing the data towards wild-type alleles and increasing false negative results for somatic mutations. With growing interest in cfDNA-based diagnostics, several solutions have emerged to streamline pre-analytical processing These include special blood collection tubes that contain cell-stabilizing preservative to minimize lysis of peripheral blood cells for up to several days after venipuncture. We use this assay to compare cfDNA extraction kits and to perform quality assessment of plasma samples across multiple archival and prospective clinical cohorts of cancer patients. We evaluate the performance and downstream effects of blood collection tubes in matched plasma samples from healthy volunteers
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
