Standardized droplet preamplification method for downstream circulating cell-free DNA analysis

Abstract

Circulating cell-free DNA (ccfDNA) can be found in blood and other biofluids and is a minimally invasive biomarker for several pathological processes. As tumors become more invasive, an increasing amount of circulating tumor DNA (ctDNA) is also shed into the peripheral circulation. Combined analysis of ccfDNA and ctDNA has demonstrated prognostic and predictive value in metastatic disease. However, localized tumors shed significantly less ccfDNA/ctDNA and accurate detection remains a technical challenge. To overcome this barrier, droplet preamplification has been used to perform robust multiplexed analysis of low input samples. To reduce false positives, it is essential to use a high-fidelity polymerase with 3-5 exonuclease activity. However, attempts to combine high-fidelity polymerases with commercial droplet digital chemistries have had limited success. There is also no standardized method for efficient amplicon recovery from droplets. In this work, we present a method to reliably stabilize emulsions and recover preamplified template. We systematically compared our protocol with different destabilization methods and found an average 41% improvement in recovery efficiency. We anticipate this standardized method will increase the consistency and reproducibility of ccfDNA/ctDNA analyses. This technique could be readily translated to other low-input or low-biomass samples, such as urine, saliva, or archived biopsy specimens.