Table 2.

Summary of standard methods.

Standard method	Overview
Collection of Normal Human Plasma	Minimize pooling of plasma from multiple donors; single donors are preferred. Collect blood in the appropriate blood collection tube (BCT) for the assay’s intended use. Process the plasma within the recommended stability window for that BCT. See also the Supplemental Material 1.
Preparation of Patient Sample Pools	Quantify the variant of interest in a patient’s total ctDNA at the DNA molecule level. Calculate the appropriate volume of each patient ctDNA and wild type (WT) ctDNA to add to pool in order to achieve the targeted MAF.a Confirm that the targeted MAFs have been reached through an orthogonal quantification step.
Preparation of Contrived Samples using ctDNA from Cell Culture Media	Extract mutant-positive ctDNA from cell culture of the appropriate cell line. Culture the cell lines and collect cell culture media as specified in Bronkhorst et al. 2016 (22). Quantify ctDNA using an orthogonal method and dilute into WT ctDNA derived from plasma from normal donors. Confirm that the targeted MAFs have been reached through an orthogonal quantification step. See also the Supplemental Material 1.
Preparation of Contrived Samples using Fragmented Cell Line Genomic DNA	Extract ctDNA from cell line cells and fragment. Quantify fragmented ctDNA and assess fragment size. Dilute into fragmented WT ctDNA extracted from plasma from normal donors. Confirm that the targeted MAFs have been reached through an orthogonal quantification step. See also the Supplemental Material 1.

^aMutant allelic fraction.