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.

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.

• Authors: Jamie J Beagan, Esther E E Drees, Phylicia Stathi, Paul P Eijk, Laura Meulenbroeks, Floortje Kessler, Jaap M Middeldorp, D Michiel Pegtel, Josée M Zijlstra, Daoud Sie, Daniëlle A M Heideman, Erik Thunnissen, Linda Smit, Daphne de Jong, Florent Mouliere, Bauke Ylstra, Margaretha G M Roemer, Erik van Dijk
• Journal: The Journal of molecular diagnostics : JMD (J Mol Diagn) Vol. 23 Issue 11 Pg. 1553-1563 (11 2021) ISSN: 1943-7811 [Electronic] United States
• PMID: 34454114 (Publication Type: Comparative Study, Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't)
• Copyright: Copyright © 2021 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Chemical References

• Biomarkers, Tumor
• Circulating Tumor DNA

Topics

• Biomarkers, Tumor (blood, genetics)
• Blood Specimen Collection (methods)
• Carcinoma, Non-Small-Cell Lung (blood, diagnosis, genetics)
• Case-Control Studies
• Circulating Tumor DNA (blood, genetics)
• DNA Copy Number Variations
• Feasibility Studies
• Humans
• Leukemia, Myeloid, Acute (blood, diagnosis, genetics)
• Limit of Detection
• Liquid Biopsy
• Longitudinal Studies
• Lung Neoplasms (blood, diagnosis, genetics)
• Lymphoma, B-Cell (blood, diagnosis, genetics)
• Polymerase Chain Reaction (methods)
• Whole Genome Sequencing (methods)