Abstract
We developed and benchmarked Exome Cancer Test v.2.0 (EXaCT-2), a novel whole-exome sequencing (WES) assay based on Agilent’s SureSelect hybrid-capture technology and expanded with custom probes targeting cancer-informative genomic regions. EXaCT-2 provides ~1,400 cancer genes with the depth of coverage typical of targeted panels, while achieving the genomic breadth to detect somatic copy number alterations (SCNAs), common cancer-related rearrangements, oncogenic viruses and B-cell receptor (BCR) clonotypes. Evaluated with a cancer patient cohort of 244 matched tumor/normal pairs and compared with clinically-validated results, EXaCT-2 achieved a mean sequencing depth of ~400× for critical cancer genes and ~100× for the remainder of the exome, with SCNA characterization showing improved boundary detection and overall segmentation. The assay demonstrated enhanced sensitivity for detecting sub-clonal, low-allele-frequency mutations missed by standard exome assays, such as mutations in GC-rich genes like KRAS. Analysis is performed by a modular, bespoke pipeline that leverages a workflow manager (Nextflow), in combination with containerized open-source tools. In addition to mutations and SCNAs, the pipeline reports common cancer rearrangements, hematologic oncogenic viruses, BCR clonotypes, and global molecular metrics, such as tumor mutational burden (TMB) and microsatellite instability (MSI). Collectively, these results establish EXaCT-2 as a comprehensive platform for integrated cancer genome profiling.
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Data availability
Mutational data is or will be made available on a private cBioPortal server. Credentials for access can and will be made available upon request of the editors. Raw, read data is in the process of being deposited to the database of Genotype and Phenotype (dbGaP).
Code availability
The source code for the pipeline used for the analysis will be made available as a private Github repository. Eventually, this will become a publicly available release. Credentials for access can and will be made available on the request of the editors. Tools and Versioning information are provided in Table S19.
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Acknowledgements
We acknowledge help from the following institutions and individuals. Giuseppe Giaccone, MD/PhD, for the GTF2I and MTAP customization discussion. Alessandro Romanel, PhD, University of Trento - Italy, for technical input and discussions. The Genomics and Epigenomics Core Research Facility at Weill Cornell Medicine for the library preparation and sequencing. The Englander Institute of Precision Medicine (EIPM) and the Joint Clinical Genomics Initiative (JCGI) of Weill Cornell Medicine and New York Presbyterian Hospital for funding this project.
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Analysis of the data, A.A.; A.S.; B.B.; J.M.M.; J.P.S.; K.E.; P.C.; P.W.; W.S.; Code review, K.G.; Data/Sample collection/provision, A.A.; H.B.; H.R.; J.M.; J.M.; J.M.M.; J.P.S.; K.E.; M.A.A.; M.M.O.; M.S.; N.L.; O.E.; W.S.; W.T.; Data/Sample processing, A.K.; D.C.W.; E.M.F.; H.J.P.; J.M.M.; K.E.; M.S.; P.C.; P.D.; P.W.; T.K.; Design of the study, A.S.; D.C.H.; D.C.W.; HR.;. M.S.; O.E.; R.K.; W.S.; Editing/reviewing the manuscript, A.A.; A.S.; C.P.; D.C.H.; D.C.W.; D.R.; F.D.; H.B.; H.J.P.; J.C.; J.M.; J.M.M.; J.P.S.; K.G.; M.A.A.; M.M.O.; M.S.; N.L.; P.W.; T.T.E.; Funding, J.C.; N.L.; O.E.; R.K.; Infrastructure, A.S.; J.T.; P.Z.; Probe and Assay Design, C.P.; D.C.H.; D.R.; F.D.; T.TE.; Testing and Verification, C.P.; J.P.S.; W.S.; Writing of the manuscript, A.A.; A.S.; D.C.W.; P.C.; P.W.
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Theresa Ten Eyck, Douglas Roberts, and Carlos Pabon are or were employees of Agilent Technologies, the maker/producer of the EXaCT-2 assay. The other authors do not have a competing interest.
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Waltman, P., Chandra, P., Eng, K.W. et al. EXaCT-2: an augmented and customizable oncology-focused whole exome sequencing platform. npj Precis. Onc. (2026). https://doi.org/10.1038/s41698-026-01390-5
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DOI: https://doi.org/10.1038/s41698-026-01390-5
