Abstract Background: Accurate characterization of chromosomal aberrations (cytogenetics) in hematologic malignancies is fundamental for proper disease diagnosis, prognosis and may inform treatment decisions. Current karyotyping and fluorescent in situ hybridization (FISH) clinical assays have important sensitivity and sample limitations and cannot detect many novel chromosomal abnormalities in cancer. We have developed a NGS-based targeted DNA and RNA sequencing assay (FoundationOne Heme)and analysis pipeline compatible with typical clinical specimens that can detect all types of variants including large chromosomal abnormalities at base-pair level. High accuracy for the detection of recurrent cytogenetic events was validated across 100 CLL and DLBCL specimens with known abnormalities Method: Adaptor-ligated sequencing libraries were captured by solution hybridization using a custom bait-set targeting 405 blood cancer-related genes by DNA-seq, and 265 frequently-rearranged genes by RNA-seq. All captured libraries were sequenced to high depth (Illumina HiSeq), averaging 498x for DNA and ∼7M on-target unique pairs for RNA, to enable highly accurate detection of base substitutions, indels, copy number alterations and gene rearrangements. Cytogenetics calls were determined using a computational method which compares germline SNP allele frequencies to model expectations, taking into account tumor purity, ploidy, and local copy number. Result: Assay performance demonstrated highly accurate detection of known cytogenetic events, including large deletions, trisomy and translocations, in 61 CLL and 39 DLBCL specimens previously characterized with CLIA-certified karyotyping and FISH assays. In the CLL cohort, 40 of 41 (98%) known positives and 172 of 172 (100%) known negatives were successfully characterized, including deletion of chr11q, chr13q, chr17p and chr12 trisomy. No false positive was reported in any of the cytogenetic variants. In the DLBCL cohort, 35 of 39 (90%) known translocations involving IGH-MYC, IGH-BCL2 and IGH-BCL6 were detected. Accurate detection of these rearrangements is particularly difficult as they are not typical gene fusions and the genomic breakpoints are highly variable, distributed across upstream, genic and downstream of the targeted genes. In addition, deletion in chr17p including TP53 was detected from 37 of 165 DLBCL patients with large length distribution ranging from 500 kb to 79 Mb. Conclusion: Accurate detection of cytogenetic events at higher resolution than traditional cytogenetics and FISH techniques is possible using an optimized NGS-based assay and tailored algorithms. An improved understanding of the molecular details of chromosomal abnormalities, integrated with comprehensive profiling of other cancer relevant genomic alterations, may help the upfront characterization of these alterations and impact diagnosis, prognosis and therapeutic intervention. Citation Format: Jie He, Jeffrey Gardner, James X. Sun, Omar Abdel-Wahab, Andrew M. Intlekofer, Michelle K. Nahas, Jo-Anne Vergilio, Jeffery S. Ross, Roman Yelenskey, Ross Levine, Geoff Otto, Doron Lipson, Vincent Miller, Philip Stephens. Next-generation sequencing enables new approach to molecular cytogenetics. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4927. doi:10.1158/1538-7445.AM2015-4927