Presentation Information
[P01-127]A robust low-pass whole-genome sequencing workflow for detecting copy number alterations in single circulating tumor cells
○Kouga Kamio1, Takatsugu Okegawa2, Mayumi Deki2, Yuu Nakamura2, Tomoko Yoshino1 (1. Division of Biotechnology and Life Science, Institute of Engineering, Tokyo University of Agriculture and Technology (Japan), 2. Department of Urology, School of Medicine, Kyorin University, Tokyo, Japan (Japan))
Keywords:
Circulating tumor cells (CTCs),Copy number alternations (CNAs),Low-pass whole genome sequencing (lp-WGS),Single-cell analysis,Liquid biopsy
[Purpose]
Copy number alterations (CNAs) are genomic abnormalities arising as chromosomal aneuploidy in tumor tissues and have been established as useful biomarkers, primarily through analyses of tissue biopsy specimens. However, the invasive nature of biopsies limits their application for disease monitoring and treatment response prediction. Circulating tumor cells (CTCs) have emerged as a minimally invasive alternative enabling longitudinal sampling, yet technical bottlenecks remain and standardized workflows are lacking. Here, we aimed to establish a robust low-pass whole-genome sequencing (lp-WGS) workflow for the reproducible detection of CNAs from single CTCs.
[Methods]
The human urothelial carcinoma cell line T24 was processed using the Microcavity Array (MCA)/Gel-based Cell Manipulation (GCM) method developed in our laboratory. Whole-genome amplification (WGA) was performed on both single-cell-isolated samples and extracted genomic DNA samples, representing WGA-based and non-WGA-based conditions. All samples were sequenced on the Illumina NovaSeq platform (2 × 150 bp) at a read depth of approximately 1×, and copy number was estimated using ichorCNA after alignment. Whole blood samples were collected from patients with urothelial carcinoma. Samples were stained with DAPI and antibodies against cytokeratin (CK) and CD45. Cells positive for CK, negative for CD45, and positive for DAPI were defined as CTCs. CTCs isolated from patient samples were processed in the same manner as the cell line samples.
[Results & Discussion]
CNA detection in three cell line-derived samples showed that WGA increased noise levels, while genome-wide CNA patterns were largely preserved. Downsampling analysis demonstrated that focal CNAs spanning 20-30 Mb were reproducible at sub-1× coverage, whereas chromosome arm level CNAs remained detectable even at substantially lower sequencing depths. These results indicate that the protocol enables robust CNA detection at the single-cell level irrespective of WGA, even under extremely low sequencing depth conditions. CNA analysis of patient-derived CTCs confirmed that CNAs could be detected from single CTCs. Although inter-patient heterogeneity was observed, recurrent copy number gains, including alterations involving chromosomes frequently implicated in urothelial carcinoma, were identified across multiple patients. Notably, genes located within these altered regions include those previously implicated in malignant transformation, suggesting that this protocol enables detection of clinically relevant genomic alterations from single CTCs.
[Conclusion]
We established a highly reproducible workflow for CNA detection by applying lp-WGS to single CTCs isolated using the MCA/GCM method. This protocol allows flexible sequencing depth design tailored to specific research needs and is expected to have broad applications, ranging from elucidation of tumor evolution to biomarker discovery.
Copy number alterations (CNAs) are genomic abnormalities arising as chromosomal aneuploidy in tumor tissues and have been established as useful biomarkers, primarily through analyses of tissue biopsy specimens. However, the invasive nature of biopsies limits their application for disease monitoring and treatment response prediction. Circulating tumor cells (CTCs) have emerged as a minimally invasive alternative enabling longitudinal sampling, yet technical bottlenecks remain and standardized workflows are lacking. Here, we aimed to establish a robust low-pass whole-genome sequencing (lp-WGS) workflow for the reproducible detection of CNAs from single CTCs.
[Methods]
The human urothelial carcinoma cell line T24 was processed using the Microcavity Array (MCA)/Gel-based Cell Manipulation (GCM) method developed in our laboratory. Whole-genome amplification (WGA) was performed on both single-cell-isolated samples and extracted genomic DNA samples, representing WGA-based and non-WGA-based conditions. All samples were sequenced on the Illumina NovaSeq platform (2 × 150 bp) at a read depth of approximately 1×, and copy number was estimated using ichorCNA after alignment. Whole blood samples were collected from patients with urothelial carcinoma. Samples were stained with DAPI and antibodies against cytokeratin (CK) and CD45. Cells positive for CK, negative for CD45, and positive for DAPI were defined as CTCs. CTCs isolated from patient samples were processed in the same manner as the cell line samples.
[Results & Discussion]
CNA detection in three cell line-derived samples showed that WGA increased noise levels, while genome-wide CNA patterns were largely preserved. Downsampling analysis demonstrated that focal CNAs spanning 20-30 Mb were reproducible at sub-1× coverage, whereas chromosome arm level CNAs remained detectable even at substantially lower sequencing depths. These results indicate that the protocol enables robust CNA detection at the single-cell level irrespective of WGA, even under extremely low sequencing depth conditions. CNA analysis of patient-derived CTCs confirmed that CNAs could be detected from single CTCs. Although inter-patient heterogeneity was observed, recurrent copy number gains, including alterations involving chromosomes frequently implicated in urothelial carcinoma, were identified across multiple patients. Notably, genes located within these altered regions include those previously implicated in malignant transformation, suggesting that this protocol enables detection of clinically relevant genomic alterations from single CTCs.
[Conclusion]
We established a highly reproducible workflow for CNA detection by applying lp-WGS to single CTCs isolated using the MCA/GCM method. This protocol allows flexible sequencing depth design tailored to specific research needs and is expected to have broad applications, ranging from elucidation of tumor evolution to biomarker discovery.
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