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

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

doi:10.1016/j.jmoldx.2021.08.008

Jamie J Beagan, Esther E.E Drees + Show 16 more

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https://doi.org/10.1016/j.jmoldx.2021.08.008

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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.

Highlights

Somatic copy number alterations can be detected in cell-free DNA by shallow whole genome sequencing
The mapping quality of sequencing data was highly comparable between the 32 EDTA samples that underwent PCR and the 35 samples that underwent PCR-free library preparations, as demonstrated by the Q37 scores (PCR method, 92.5%; SD, 0.36%; PCR-free method, 93.1%; SD, 0.30%), and the percentage of reads aligned to the genome (PCR method, 99.1%; SD, 0.36%; PCR-free method, 99.1%; SD, 0.27%)
The G-C base pair content of aligned sequencing reads was highly comparable between the 32 EDTA samples that underwent PCR library preparations and the 35 samples that underwent PCR-free library preparations, which indicates that the omission of PCR does not affect the genome-wide base pair representation (Figure 2B)

Summary

Introduction

Somatic copy number alterations can be detected in cell-free DNA (cfDNA) by shallow whole genome sequencing (sWGS). 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. 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-heparinecontaining tubes and pave the way for an automated cfDNA analysis workflow for samples from cancer patients. Somatic copy number alterations (SCNAs) drive a wide range of cancer types and are utilized as molecular markers.[1,2] Next to mutation analysis, specific chromosomal.

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