PCR-free shallow whole genome sequencing for chromosomal copy number detection from plasma of cancer patients is an efficient alternative to the conventional PCR-based approach.

Somatic copy number alterations (SCNAs) can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). Polymerase chain reaction (PCR) is typically included in library preparations but a PCR-free method could serve as a high throughput alternative. To evaluate a PCR-free method for research and diagnostics, archival peripheral blood or bone marrow plasma samples collected in EDTA- or lithium heparin-containing tubes were collected from patients with: non-small cell lung cancer (n=10 longitudinal samples; 4 patients), B-cell lymphoma (n=31), acute myeloid leukemia (n=15) or from healthy donors (n=14). sWGS was performed on PCR-free and PCR library preparations and the mapping quality, percentage of unique reads, genome coverage, fragment lengths and copy number profiles were compared. The percentage of unique reads was significantly higher for PCR-free compared to PCR, independent of the type of collection tube; EDTA PCR-free: 96.4%, n=35; EDTA PCR: 85.1%, n=32; heparin PCR-free: 94.5%, n=25; heparin PCR: 89.4%, n=10. All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of SCNA detection by PCR-free sWGS using cfDNA from plasma collected in EDTA- or lithium heparin-containing tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients.
Copyright © 2021. Published by Elsevier Inc.