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PMS2 is one of the DNA-mismatch repair genes included in routine genetic testing for Lynch syndrome and colorectal, ovarian, and endometrial cancers. PMS2 is also included in the American College of Medical Genetics and Genomics' List of Secondary Findings Genes in the context of clinical exome and genome sequencing. However, sequencing of PMS2 by short-read-based next-generation sequencing technologies is complicated by the presence of the pseudogene PMS2CL, and is often supplemented by long-range-based approaches, such as long-range PCR or long-read-based next-generation sequencing, which increases the complexity and cost. This article describes a bioinformatics homology triage workflow that can eliminate the need for long-read-based testing for PMS2 in the vast majority of patients undergoing exome sequencing, thus simplifying PMS2 testing and reducing the associated cost., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Lymphoid malignancies are a heterogeneous group of hematological disorders characterized by a diverse range of morphologic, immunophenotypic, and clinical features. Next-generation sequencing (NGS) is increasingly being applied to delineate the complex nature of these malignancies and identify high-value biomarkers with diagnostic, prognostic, or therapeutic benefit. However, there are various challenges in using NGS routinely to characterize lymphoid malignancies, including pre-analytic issues, such as sequencing DNA from formalin-fixed, paraffin-embedded tissue, and optimizing the bioinformatic workflow for accurate variant calling and filtering. This study reports the clinical validation of a custom capture-based NGS panel to test for molecular markers in a range of lymphoproliferative diseases and histiocytic neoplasms. The fully validated clinical assay represents an accurate and sensitive tool for detection of single-nucleotide variants and small insertion/deletion events to facilitate the characterization and management of patients with hematologic cancers specifically of lymphoid origin., Competing Interests: Disclosure Statement None declared., (Copyright © 2024 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Innovation in sequencing instrumentation is increasing the per-batch data volumes and decreasing the per-base costs. Multiplexed chemistry protocols after the addition of index tags have further contributed to efficient and cost-effective sequencer utilization. With these pooled processing strategies, however, comes an increased risk of sample contamination. Sample contamination poses a risk of missing critical variants in a patient sample or wrongly reporting variants derived from the contaminant, which are particularly relevant issues in oncology specimen testing in which low variant allele frequencies have clinical relevance. Small custom-targeted next-generation sequencing (NGS) panels yield limited variants and pose challenges in delineating true somatic variants versus contamination calls. A number of popular contamination identification tools have the ability to perform well in whole-genome/exome sequencing data; however, in smaller gene panels, there are fewer variant candidates for the tools to perform accurately. To prevent clinical reporting of potentially contaminated samples in small next-generation sequencing panels, we have developed MICon (Microhaplotype Contamination detection), a novel contamination detection model that uses microhaplotype site variant allele frequencies. In a heterogeneous hold-out test cohort of 210 samples, the model displayed state-of-the-art performance with an area under the receiver-operating characteristic curve of 0.995., (Copyright © 2023 Association for Molecular Pathology and American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)