Abstract
UltraPrep is an open-source, two-step method for purification of cell-free DNA that entails extraction of total DNA followed by size-selective enrichment of the smaller fragments that are characteristic of DNA originating from fragmentation between nucleosome. The advantages of the two related protocols that are described are that they can easily accommodate a wide range of sample input volumes, they rely on simple, magnetic bead-based technology, the yields of cfDNA are directly comparable to the most popular methods for cfDNA purification, and they dramatically reduce the cost of cfDNA isolation relative to currently available commercial methods. We provide a framework for physical and molecular quality analysis of purified cfDNA and demonstrate that the cfDNA generated by UltraPrep meets or exceeds the quality metrics of the most commonly used procedure. In addition, our method removes high molecular weight genomic DNA (hmwgDNA) that can interfere with downstream assay results, thereby addressing one of the primary concerns for preanalytical collection of blood samples.
Highlights
Circulating cell-free DNA has shown tremendous utility as an analyte in prenatal genetic analysis and in precision medicine approaches to diagnosed cancers
Magnetic beads are one of the most expensive components in the process and we found they are most effective when added in amounts proportional to volume
The method is scalable, meaning the yield of extracted DNA per milliliter of input is consistent across a broad range of sample input volumes
Summary
Circulating cell-free DNA (cfDNA) has shown tremendous utility as an analyte in prenatal genetic analysis and in precision medicine approaches to diagnosed cancers. It holds promise to contribute to early detection of solid tumors [1,2]. This analyte has shown potential for the rapid detection of infectious microbes [3]. Detection tests that use cfDNA must be both highly sensitive and specific. Straightforward probability and statistics considerations indicate that this requires high input levels of cfDNA and subsequent genomic analysis that covers several thousand independent cfDNA “genome equivalents” [4,5]. In addition to individual patient testing, there is a largely unmet need for large, well-characterized, single-donor lots of normal human cfDNA that can be used for diagnostic test research, assay development, and routine proficiency qualification in clinical laboratory environments
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