cfDNA Input Recommendations

The Illumina Cell-Free DNA Prep with Enrichment assay requires 10 ng to 30 ng cfDNA input. The recommended input is 20 ng. Inputs lower than 10 ng or greater than 30 ng can affect assay performance. Quantify the input nucleic acids before beginning the protocol. Follow these guidelines for input:

• Use a nucleic acid isolation method that produces high recovery yields, minimizes sample consumption, and preserves sample integrity. Column-based isolation methods such as QIAGEN QIAamp Circulating Nucleic Acid Kit and Zymo Quick-cfDNA Serum and Plasma Kit provide high yield cfDNA.
• Use a size-based quantification method such as the Agilent Fragment Analyzer System or TapeStation.

Fluorometric assays are not recommended. The presence of high molecular weight DNA can lead to an overestimation of cfDNA concentrations.

• To ensure optimal nucleic acid input, quantify the mononucleosomal peak (approximately 75 bp - 250 bp) of the cfDNA fraction.

For information on sequencing compatibility and run settings, refer to the Illumina Cell-Free DNA Prep with Enrichment support page on the Illumina support website.

Use reference materials with known variant composition, such as SeraSeq ctDNA Complete Mutation Mix.