Overall schematic and Healthy Cohort’s comparative performance

a. Schematic illustration of the experimental design across two cohorts. The Healthy Cohort was a controlled experiment where plasma samples from healthy volunteer donors were collected freshly in EDTA, Streck, and heparin separators. The Hospital Cohort plasma samples were retrospectively collected in EDTA and heparin separators as part of patient care and leftover from routine clinical testing. The Hospital Cohort samples underwent an initial soft spin during the routine clinical workflow and then a hard spin after refrigerated storage. Processed samples were analyzed using whole-genome sequencing (WGS) and/or FLEXseq methylation testing. b. Healthy Cohort fragment size distribution of cfDNA collected across the three tubes. c. Healthy Cohort comparison of end motif rankings in heparin separator samples versus EDTA or Streck tubes. d. Healthy Cohort heatmap and correlation of methylation beta values in paired samples from a healthy donor (P140). Heparin separators (y-axis) are compared to EDTA and Streck tubes (x-axis). e. Healthy Cohort estimation of cell-type proportions using methylation deconvolution for each collection tube.

Viral load correlation of the Hospital Cohort

Correlation of plasma viral DNA reads detected by whole genome sequencing in paired cases collected in EDTA and heparin separators. Samples were collected at two institutions (Stanford - blue, UCSF - red, EBV - circle, HHV6 - triangle, other virus - square). RPM: reads per million human-aligned reads.

Copy number analysis of the Hospital Cohort

a. An example of genome-wide copy number profiles from Patient 34. Case P171 was collected in a heparin separator; Case P187 was collected in EDTA tube. b. Comparison of log2 copy ratios between P171 and P187 based on 500 kb bin sizes. c. Comparison of tumor fraction in paired cases (n=28 pairs) inferred from copy number analysis.

Methylation correlation and cell type deconvolution analysis in the Hospital Cohort

a. Heatmap showing methylation beta values in paired samples (Patient 31, P185 versus P168). b. Estimated cell-type proportions in the paired samples (P185 versus P168).

Correlation of DNA quantity

a. Scatter plot of the normalized extracted DNA concentration based on Qubit reading. Data is from 20 individuals of the Hospital Cohort and 5 individuals from the Healthy Cohort using heparin separators and EDTA tubes. Normalized DNA concentration (ng/mL plasma) in heparin separator samples (y-axis) vs. EDTA tube samples (x-axis), quantified by dsDNA Qubit and normalized to the input volume of plasma. b. Scatter plot of the log-scale normalized concentration (Qubit readout) in the heparin separators (y-axis) vs. the EDTA tube samples (x-axis). c. Scatter plot of the ratio of human reads to a constant lambda phage DNA spike-in detected in the heparin separator sample (y-axis) vs. the EDTA tube samples. This spike-in measurement is proportional to the addressable DNA quantity of the sample.

Healthy Cohort methylation correlation and cell type deconvolution

Heatmap and correlation of methylation beta values with cell type deconvolution results in paired samples from five healthy donors.

Healthy Cohort size distribution of cfDNA in EDTA and heparin tubes over time mimicking the clinical workflow

a) Schematic of the time-lapsed controlled experiment mimicking the clinical workflow. b) The fragment size distribution of cfDNA across all conditions. “0 min” represents immediate centrifugation, while “1 h” indicates samples stored at 4°C for 1 hour before the soft spin and for 4 days at 4°C before the hard spin.

Copy number analysis across Hospital Cohort cases

Comparison of other CNA-positive profiles of paired cases in the Hospital Cohort (n=5).

Methylation correlation across Hospital Cohort cases

Heatmaps of methylation beta values in paired samples collected from 11 virus-positive patients in the Hospital Cohort. One additional case is shown in Figure 4.

Fragmentomic patterns of the Hospital Cohort

a. Average cfDNA fragment size distribution collected in EDTA tubes and heparin separators. b. Average end motif frequency comparison between EDTA tubes and heparin separators, highlighting the top six motifs: CCCA, CCAG, CCTG, CCCT, CCAT, and CAAA.