Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

Li-Ting Chen; Myrthe Jager; Dàmi Rebergen; Geertruid J Brink; Tom van den Ende; Willem Vanderlinden; Pauline Kolbeck; Marc Pagès-Gallego; Ymke van der Pol; Nicolle Besselink; Norbert Moldovan; Nizar Hami; Wigard P Kloosterman; Hanneke van Laarhoven; Florent Mouliere; Ronald Zweemer; Jan Lipfert; Sarah Derks; Alessio Marcozzi; Jeroen de Ridder

doi:10.1101/gr.279144.124

Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

Li-Ting Chen, Myrthe Jager, Dàmi Rebergen, Geertruid J Brink, Tom van den Ende, Willem Vanderlinden, Pauline Kolbeck, Marc Pagès-Gallego, Ymke van der Pol, Nicolle Besselink, Norbert Moldovan, Nizar Hami, Wigard P Kloosterman, Hanneke van Laarhoven, Florent Mouliere, Ronald Zweemer, Jan Lipfert, Sarah Derks, Alessio Marcozzi, Jeroen de Ridder^*

^*Corresponding author for this work

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Abstract

Shallow genome-wide cell-free DNA sequencing holds great promise for noninvasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single-nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here, we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction (TF) estimation through SNVs, CNAs, and fragmentomics. The efficacy of NanoRCS is tested on 18 cancer patient samples and seven healthy controls, demonstrating its ability to reliably detect TFs as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting TFs below 3%. NanoRCS provides an opportunity for cost-effective and rapid sample processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.

Original language	English
Pages (from-to)	886-899
Number of pages	14
Journal	Genome Research
Volume	35
Issue number	4
Early online date	13 Jan 2025
DOIs	https://doi.org/10.1101/gr.279144.124
Publication status	Published - 14 Apr 2025

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Genome Res.-2025-Chen-886-99Final published version, 4.3 MBLicence: CC BY

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Chen, L.-T., Jager, M., Rebergen, D., Brink, G. J., van den Ende, T., Vanderlinden, W., Kolbeck, P., Pagès-Gallego, M., van der Pol, Y., Besselink, N., Moldovan, N., Hami, N., Kloosterman, W. P., van Laarhoven, H., Mouliere, F., Zweemer, R., Lipfert, J., Derks, S., Marcozzi, A., & de Ridder, J. (2025). Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling. Genome Research, 35(4), 886-899. https://doi.org/10.1101/gr.279144.124

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title = "Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling",

abstract = "Shallow genome-wide cell-free DNA sequencing holds great promise for noninvasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single-nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here, we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction (TF) estimation through SNVs, CNAs, and fragmentomics. The efficacy of NanoRCS is tested on 18 cancer patient samples and seven healthy controls, demonstrating its ability to reliably detect TFs as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting TFs below 3%. NanoRCS provides an opportunity for cost-effective and rapid sample processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.",

author = "Li-Ting Chen and Myrthe Jager and D{\`a}mi Rebergen and Brink, {Geertruid J} and {van den Ende}, Tom and Willem Vanderlinden and Pauline Kolbeck and Marc Pag{\`e}s-Gallego and {van der Pol}, Ymke and Nicolle Besselink and Norbert Moldovan and Nizar Hami and Kloosterman, {Wigard P} and {van Laarhoven}, Hanneke and Florent Mouliere and Ronald Zweemer and Jan Lipfert and Sarah Derks and Alessio Marcozzi and {de Ridder}, Jeroen",

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Chen, L-T, Jager, M, Rebergen, D, Brink, GJ, van den Ende, T, Vanderlinden, W, Kolbeck, P, Pagès-Gallego, M, van der Pol, Y, Besselink, N, Moldovan, N, Hami, N, Kloosterman, WP, van Laarhoven, H, Mouliere, F, Zweemer, R, Lipfert, J, Derks, S, Marcozzi, A & de Ridder, J 2025, 'Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling', Genome Research, vol. 35, no. 4, pp. 886-899. https://doi.org/10.1101/gr.279144.124

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AU - Chen, Li-Ting

AU - Jager, Myrthe

AU - Rebergen, Dàmi

AU - Brink, Geertruid J

AU - van den Ende, Tom

AU - Vanderlinden, Willem

AU - Kolbeck, Pauline

AU - Pagès-Gallego, Marc

AU - van der Pol, Ymke

AU - Besselink, Nicolle

AU - Moldovan, Norbert

AU - Hami, Nizar

AU - Kloosterman, Wigard P

AU - van Laarhoven, Hanneke

AU - Mouliere, Florent

AU - Zweemer, Ronald

AU - Lipfert, Jan

AU - Derks, Sarah

AU - Marcozzi, Alessio

AU - de Ridder, Jeroen

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N2 - Shallow genome-wide cell-free DNA sequencing holds great promise for noninvasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single-nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here, we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction (TF) estimation through SNVs, CNAs, and fragmentomics. The efficacy of NanoRCS is tested on 18 cancer patient samples and seven healthy controls, demonstrating its ability to reliably detect TFs as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting TFs below 3%. NanoRCS provides an opportunity for cost-effective and rapid sample processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.

AB - Shallow genome-wide cell-free DNA sequencing holds great promise for noninvasive cancer monitoring by providing reliable copy number alteration (CNA) and fragmentomic profiles. Single-nucleotide variations (SNVs) are, however, much harder to identify with low sequencing depth due to sequencing errors. Here, we present Nanopore Rolling Circle Amplification (RCA)-enhanced Consensus Sequencing (NanoRCS), which leverages RCA and consensus calling based on genome-wide long-read nanopore sequencing to enable simultaneous multimodal tumor fraction (TF) estimation through SNVs, CNAs, and fragmentomics. The efficacy of NanoRCS is tested on 18 cancer patient samples and seven healthy controls, demonstrating its ability to reliably detect TFs as low as 0.24%. In vitro experiments confirm that SNV measurements are essential for detecting TFs below 3%. NanoRCS provides an opportunity for cost-effective and rapid sample processing, which aligns well with clinical needs, particularly in settings where quick and accurate cancer monitoring is essential for personalized treatment strategies.

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DO - 10.1101/gr.279144.124

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VL - 35

SP - 886

EP - 899

JO - Genome Research

JF - Genome Research

IS - 4

ER -

Nanopore-based consensus sequencing enables accurate multimodal tumor cell-free DNA profiling

Abstract

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