Dose-Related Mutagenic and Clastogenic Effects of Benzo[]fluoranthene in Mouse Somatic Tissues Detected by Duplex Sequencing and the Micronucleus Assay.

Polycyclic aromatic hydrocarbons (PAHs) are common environmental pollutants that originate from the incomplete combustion of organic materials. We investigated the clastogenicity and mutagenicity of benzo[]fluoranthene (BbF), one of 16 priority PAHs, in MutaMouse males after a 28 day oral exposure. BbF causes robust dose-dependent increases in micronucleus frequency in peripheral blood, indicative of chromosome damage.

Error-corrected next generation sequencing - Promises and challenges for genotoxicity and cancer risk assessment.

Error-corrected Next Generation Sequencing (ecNGS) is rapidly emerging as a valuable, highly sensitive and accurate method for detecting and characterizing mutations in any cell type, tissue or organism from which DNA can be isolated. Recent mutagenicity and carcinogenicity studies have used ecNGS to quantify drug-/chemical-induced mutations and mutational spectra associated with cancer risk. ecNGS has potential applications in genotoxicity assessment as a new readout for traditional models, for mutagenesis studies in 3D organotypic cultures, and for detecting off-target effects of gene editing tools.

Quantification of measurable residual disease using duplex sequencing in adults with acute myeloid leukemia.

The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials and discrepancies have been observed between different techniques for MRD assessment. In 62 patients with AML, aged 18-60 years, in first complete remission after intensive induction therapy on the randomized phase III SWOG-S0106 clinical trial (clinicaltrials gov.

Error-corrected duplex sequencing enables direct detection and quantification of mutations in human TK6 cells with strong inter-laboratory consistency.

Error-corrected duplex sequencing (DS) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DS to quantify induced mutation frequency (MF) and spectrum in human lymphoblastoid TK6 cells exposed to a prototypical DNA alkylating agent, N-ethyl-N-nitrosourea (ENU). Furthermore, we explored appropriate experimental parameters for this application, and assessed inter-laboratory reproducibility.

Duplex sequencing provides detailed characterization of mutation frequencies and spectra in the bone marrow of MutaMouse males exposed to procarbazine hydrochloride.

Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data.

svCapture: efficient and specific detection of very low frequency structural variant junctions by error-minimized capture sequencing.

Error-corrected sequencing of genomic targets enriched by probe-based capture has become a standard approach for detecting single-nucleotide variants (SNVs) and small insertion/deletions (indels) present at very low variant allele frequencies. Less attention has been given to comparable strategies for rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from samples with known SV properties, we demonstrate that duplex sequencing (DuplexSeq), which demands confirmation of variants on both strands of a source DNA molecule, eliminates false SV junctions arising from chimeric PCR.

Quantification of measurable residual disease using duplex sequencing in adults with acute myeloid leukemia.

The presence of measurable residual disease (MRD) is strongly associated with treatment outcomes in acute myeloid leukemia (AML). Despite the correlation with clinical outcomes, MRD assessment has yet to be standardized or routinely incorporated into clinical trials. Discrepancies have been observed between different techniques for MRD assessment and there remains a need to compare centralized, high-quality multiparametric flow cytometry (MFC) and ultrasensitive next-generation sequencing (NGS) in AML patients with diverse mutational profiles.

Duplex sequencing identifies genomic features that determine susceptibility to benzo(a)pyrene-induced in vivo mutations.

Exposure to environmental mutagens increases the risk of cancer and genetic disorders. We used Duplex Sequencing (DS), a high-accuracy error-corrected sequencing technology, to analyze mutation induction across twenty 2.4 kb intergenic and genic targets in the bone marrow of MutaMouse males exposed to benzo(a)pyrene (BaP), a widespread environmental pollutant.

PolyG-DS: An ultrasensitive polyguanine tract-profiling method to detect clonal expansions and trace cell lineage.

Polyguanine tracts (PolyGs) are short guanine homopolymer repeats that are prone to accumulating mutations when cells divide. This feature makes them especially suitable for cell lineage tracing, which has been exploited to detect and characterize precancerous and cancerous somatic evolution. PolyG genotyping, however, is challenging because of the inherent biochemical difficulties in amplifying and sequencing repetitive regions.

Direct quantification of in vivo mutagenesis and carcinogenesis using duplex sequencing.

The ability to accurately measure mutations is critical for basic research and identifying potential drug and chemical carcinogens. Current methods for in vivo quantification of mutagenesis are limited because they rely on transgenic rodent systems that are low-throughput, expensive, prolonged, and do not fully represent other species such as humans. Next-generation sequencing (NGS) is a conceptually attractive alternative for detecting mutations in the DNA of any organism; however, the limit of resolution for standard NGS is poor.

Ultra-accurate Duplex Sequencing for the assessment of pretreatment ABL1 kinase domain mutations in Ph+ ALL.

Mutations of ABL1 are the dominant mechanism of relapse in Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph + ALL). We performed highly accurate Duplex Sequencing of exons 4-10 of ABL1 on bone marrow or peripheral blood samples from 63 adult patients with previously untreated Ph + ALL who received induction with intensive chemotherapy plus a BCR-ABL1 TKI. We identified ABL1 mutations prior to BCR-ABL1 TKI exposure in 78% of patients.

Ultra-Sensitive TP53 Sequencing for Cancer Detection Reveals Progressive Clonal Selection in Normal Tissue over a Century of Human Lifespan.

High-accuracy next-generation DNA sequencing promises a paradigm shift in early cancer detection by enabling the identification of mutant cancer molecules in minimally invasive body fluid samples. We demonstrate 80% sensitivity for ovarian cancer detection using ultra-accurate Duplex Sequencing to identify TP53 mutations in uterine lavage. However, in addition to tumor DNA, we also detect low-frequency TP53 mutations in nearly all lavages from women with and without cancer.

Enhancing the accuracy of next-generation sequencing for detecting rare and subclonal mutations.

Mutations, the fuel of evolution, are first manifested as rare DNA changes within a population of cells. Although next-generation sequencing (NGS) technologies have revolutionized the study of genomic variation between species and individual organisms, most have limited ability to accurately detect and quantify rare variants among the different genome copies in heterogeneous mixtures of cells or molecules. We describe the technical challenges in characterizing subclonal variants using conventional NGS protocols and the recent development of error correction strategies, both computational and experimental, including consensus sequencing of single DNA molecules.

Precancer in ulcerative colitis: the role of the field effect and its clinical implications.

Cumulative evidence indicates that a significant proportion of cancer evolution may occur before the development of histological abnormalities. While recent improvements in DNA sequencing technology have begun to reveal the presence of these early preneoplastic clones, the concept of 'premalignant field' was already introduced by Slaughter more than half a century ago. Also referred to as 'field effect', 'field defect' or 'field cancerization', these terms describe the phenomenon by which molecular alterations develop in normal-appearing tissue and expand to form premalignant patches with the potential to progress to dysplasia and cancer.

Exploring the implications of distinct mutational signatures and mutation rates in aging and cancer.

Signatures of mutagenesis provide a powerful tool for dissecting the role of somatic mutations in both normal and pathological processes. Significantly, cancer genomes are dominated by mutation signatures distinct from those that accumulate in normal tissues with age, with potentially important translational implications.