1. 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
- Author
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D. Michiel Pegtel, Erik van Dijk, Paul P. Eijk, Linda Smit, Margaretha G. M. Roemer, Phylicia Stathi, Erik Thunnissen, Floortje Kessler, Laura Meulenbroeks, Josee M. Zijlstra, Daphne de Jong, Bauke Ylstra, Jaap M. Middeldorp, Daniëlle A.M. Heideman, Jamie J. Beagan, Florent Mouliere, Daoud Sie, Esther E.E. Drees, Pathology, AII - Cancer immunology, CCA - Imaging and biomarkers, CCA - Cancer biology and immunology, Hematology, Human genetics, and Hematology laboratory
- Subjects
Lung Neoplasms ,Lymphoma, B-Cell ,DNA Copy Number Variations ,Biology ,Polymerase Chain Reaction ,Genome ,Circulating Tumor DNA ,Pathology and Forensic Medicine ,chemistry.chemical_compound ,Copy Number Alteration ,Limit of Detection ,Carcinoma, Non-Small-Cell Lung ,Biomarkers, Tumor ,medicine ,Humans ,Longitudinal Studies ,Whole genome sequencing ,Blood Specimen Collection ,Whole Genome Sequencing ,Liquid Biopsy ,Cancer ,Myeloid leukemia ,medicine.disease ,Molecular biology ,Peripheral blood ,Leukemia, Myeloid, Acute ,medicine.anatomical_structure ,chemistry ,Case-Control Studies ,Feasibility Studies ,Molecular Medicine ,Bone marrow ,DNA - Abstract
Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). 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), and 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 method compared with PCR method, independent of the type of collection tube: EDTA PCR-free method, 96.4% (n = 35); EDTA PCR method, 85.1% (n = 32); heparin PCR-free method, 94.5% (n = 25); and heparin PCR method, 89.4% (n = 10). All other evaluated metrics were highly comparable for PCR-free and PCR library preparations. These results demonstrate the feasibility of somatic copy number alteration 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.
- Published
- 2021