Optimized Library Prep for Cell-Free DNA from Human Plasma

Researchers and clinicians are increasingly interested in using Next Generation Sequencing (NGS) analysis of cell-free DNA (cfDNA) found in plasma (the cell-free fraction of anticoagulated blood) for biomarker discovery and diagnostic applications. Two areas of specific interest are non-invasive prenatal diagnostics (1-6) and monitoring efficacy of treatment in cancer patients (7-20). Many recent studies have shown the feasibility of detecting fetal aneuploidies such as Chromosome 21 trisomy (the cause of Down’s Syndrome) by shotgun sequencing of DNA-Seq libraries produced from cell-free DNA isolated from maternal blood. Other aneuploidies including trisomies of Chromosomes 13 and 18 have also been detected (3). This approach is attractive since it avoids the risk of miscarriage associated with invasive tests (amniocentesis and chorionic villi sampling) and may offer cost benefits for prenatal diagnosis. Also, NGSbased assessment has the potential to uncover defects arising from more subtle genetic alterations including point mutations, insertion/ deletion mutations, translocations, etc. Although intact fetal cells can be detected in maternal blood, they are vastly outnumbered by the mother’s blood cells, and the proportional concentration of informative fetal DNA sequences has been reported to be higher in cell-free circulating DNA in plasma compared to DNA recovered from whole blood. The same findings hold true in the case of cancer diagnostics, where the genetic signal from rare circulating tumor cells is harder to discern against the background of non-malignant cells in whole blood, compared to circulating cell-free DNA. Assessing cancer-related genetic alterations in cell-free DNA can also avoid positional bias inherent in direct sampling of tumors, where the spectrum of mutations observed can differ for different biopsy locations within the malignant tissue. The concept of “liquid biopsy” refers to using readily obtainable body fluids, primarily blood and blood fractions, as surrogate tissue for monitoring levels of malignancy-associated mutations that arise from tumor cells and cell-free DNA shed from tumors (18 20). Monitoring levels of cancer-associated genetic alterations in cell-free DNA in blood plasma is a promising new approach to assess the benefits of chemotherapy and other types of cancer treatment. Liquid biopsy of circulating cell-free DNA also has potential for early detection of cancer, and for stratifying cancer patients for treatment decisions. To realize the full potential of these promising opportunities, robust and standardized methods are needed for creating DNA-Seq libraries from cell-free DNA extracted from plasma. To meet this need, Bioo Scientific has developed the NEXTflexTM Cell Free DNA-Seq Kit designed for making NGS libraries from low-input samples. Below, we report results demonstrating the use of this kit to produce high-quality informative libraries from cell-free DNA extracted from plasma.