7028 Background: Hematological neoplasms are often characterized by acute onset and rapid disease progression. Cytogenetics, FISH, SNP arrays, targeted DNA and RNA sequencing are performed to inform diagnosis, risk stratification and guide treatment decisions. Whole genome sequencing (WGS) offers the opportunity to comprehensively characterize all putative biomarkers in a single assay. However, a limitation in current WGS implementation is the requirement for a germline sample, as sources of control tissue are frequently contaminated with leukemic cells resulting in false negative calls. Methods: To evaluate the clinical utility and feasibility of WGS in the diagnostic work up of leukemias, we analyzed 57 B-cell acute lymphoblastic leukemia (B-ALL) from the UKALL14 trial (NCT01085617) with no informative biomarkers at diagnosis. WGS analysis was performed on the leukemic sample and a matching control sample (with minimal residual disease level of <1%). Using this dataset, we trained the development of an unmatched (uWGS) analytical workflow (Isabl) for a tumor only WGS study. This workflow was validated across 20 hematologic neoplasms (12 B-ALL, 6 AML and 2 T-ALL). Results: Among the 57 cases, 5 failed QC owing to low tumor content (<25%). Of the remaining 52, putative biomarkers of clinical relevance were identified by WGS in 69% (36/52). These included delineation of aberrant karyotypes where conventional chromosome banding failed (4/52), the detection of newly described fusion genes (such as IGH-DUX4 and EP300-ZNF384 in 21/52) and recurrent gene mutations (i.e. PAX5 P80R, ZEB2 H1038R in 11/52). uWGS workflow in our training dataset captured 86% of biomarkers identified in the matched analysis (3/3 ploidy, 21/22 fusion and 7/11 coding). Concordance between the matched and uWGS workflow for arm-level and focal copy number alterations (CNAs), structural variants (SVs) and annotated hotspot mutations were 94%, 84%, 83% and 100% respectively. Independent validation of the uWGS workflow across 20 myeloid and lymphoid neoplasms, recapitulated all clinically reported biomarkers (14/15 CNAs, 16/16 SVs) as well as captured two novel findings not previously detected in two B-ALL patients, to include a focal deletion in BTG1 and the fusion gene P2RY8-CRLF2, as well as a NOTCH1 translocation in T-ALL. Conclusions: Our findings demonstrate that comprehensive WGS allows for the detection of the same biomarkers as a range of clinical assays using a single test, as well as the opportunity to discover novel clinical and research findings to support future correlative research and biomarker development. Additionally, we developed and validated an uWGS workflow that allows WGS analysis of hematopoietic neoplasms at diagnosis, enabling detection and reporting of clinically relevant biomarkers.