Biology subjects

Salk, J. J.

Publications and source records attributed to Salk, J. J..

11 recordsLinked to original sources

bioRxiv2026

Ultra-deep duplex sequencing reveals unique features of somatic evolution in the normal tissues of a family with Li-Fraumeni syndrome

Li-Fraumeni Syndrome (LFS) is caused by germline pathogenic variants in TP53 which predispose carriers to early onset cancer across multiple tissues. While genomically profiling those cancers has revealed factors contributing to their formation, little is understood about how LFS impacts clonal evolution in healthy tissues preceding cancer. Here, we use ultra-deep duplex sequencing (mean [~]15,000x depth) to investigate somatic mutation and selection in a family carrying the germline TP53 p.R181H pathogenic variant and a cohort of non-carrier controls. In blood samples, the germline variant is associated with more mutations in a panel designed to capture genomewide mutagenesis, and with reduced positive selection on somatic TP53 mutations, despite confounding by chemotherapy treatment in one individual. DNMT3A and TET2 mutations appear positively selected and GATA2 mutations negatively selected across the cohort, independent of the p.R181H status. Extensive multi-tissue sampling of 22 non-cancerous and 6 cancerous samples was also performed at autopsy in one individual with LFS who succumbed to esophageal cancer. Cross-tissue analysis reveals excess mutations in sun-exposed skin, esophagus and chronically-inflamed stomach tissue, and concordant mutations in the p.R248 hotspot of TP53 across most (18/28) tissue samples. Most somatic TP53 mutations in LFS that can be assessed for phase arose on the chromosomal copy lacking the p.R181H variant. Our study reveals how the germline p.R181H variant reshapes baseline somatic mutation and selection in normal tissues and highlights the importance of understanding early somatic evolution in LFS prior to cancer development and treatment.

cancer biology↗

bioRxiv2025

Alignment between Duplex Sequencing and transgenic rodent mutation assay data in the assessment of in vivo NDMA-induced mutagenesis

The nitrosamine N-nitrosodimethylamine (NDMA) is a mutagen and rodent carcinogen that has been identified as a process impurity in some commercially available medicines, leading to market withdrawals and new impurity control measures. Error-corrected DNA sequencing techniques, such as Duplex Sequencing (DS), have error rates low enough to revolutionise genetic toxicology testing by directly measuring in vivo mutagenesis within days of exposure. Here, DS was performed on liver samples from an OECD-compliant, Transgenic Rodent Gene Mutation Assay (TGR) conducted under GLP standards. MutaMouse specimens were orally dosed with NDMA using either a repeat-dose 28-day regimen (0.02-4 mg/kg(bw)/day) or single bolus doses of either 5 or 10 mg/kg(bw) administered on day one. Dose-dependent increases in mutation frequency were detected by DS in liver, enabling a No-Observed Genotoxic Effect Level (NOGEL) of 0.07 mg/kg(bw)/day to be determined, supported by mechanistic analyses of trinucleotide mutation spectra. Benchmark dose (BMD) modelling determined similar BMD50 values from either DS or TGR, demonstrating concordance across the two techniques albeit with greater precision from DS due to smaller inter-animal variation. DS offers a fundamental change in mutagenicity assessments enabling more precise point-of-departure determinations with mechanistic clarity and 3Rs advantages compared to the standard TGR approach.

pharmacology and toxicology↗

bioRxiv2024

Dose-related Mutagenic and Clastogenic Effects of Benzofluoranthene 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[b]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. Duplex Sequencing (DS), an error-corrected sequencing technology, reveals that BbF induces dose-dependent increases in mutation frequencies in bone marrow (BM) and liver. Mutagenicity is increased in intergenic relative to genic regions, suggesting a role for transcription-coupled repair of BbF-induced DNA damage. At higher doses, the maximum mutagenic response to BbF is higher in liver, which has a lower mitotic index but higher metabolic capacity than BM; however, mutagenic potency is comparable between the two tissues. BbF induces primarily C:G>A:T mutations, followed by C:G>T:A and C:G>G:C, indicating that BbF metabolites mainly target guanines and cytosines. The mutation spectrum of BbF correlates with cancer mutational signatures associated with tobacco exposure, supporting its contribution to the carcinogenicity of combustion-derived PAHs in humans. Overall, BbFs mutagenic effects are similar to benzo[a]pyrene, a well-studied mutagenic PAH. Our work showcases the utility of DS for effective mutagenicity assessment of environmental pollutants. SynopsisWe used Duplex Sequencing to study the mutagenicity of benzo[b]fluoranthene across the mouse genome. Dose-dependent changes in mutation frequency and spectrum quantify its role in PAH-induced carcinogenicity.

pharmacology and toxicology↗

bioRxiv2023

Adopting Duplex Sequencing Technology for Genetic Toxicity Testing: A Proof-of-Concept Mutagenesis Experiment with N-Ethyl-N-Nitrosourea (ENU)-Exposed Rats

Duplex sequencing (DuplexSeq) is an error-corrected next-generation sequencing (ecNGS) method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors by comparing grouped strand sequencing reads. The resulting background of less than one artifactual mutation per 107 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DuplexSeq-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues, a considerable advancement compared to currently used in vivo gene mutation assays. HIGHLIGHTSO_LIDuplexSeq is an ultra-accurate NGS technology that directly quantifies mutations C_LIO_LIENU-dependent mutagenesis was detected 24 h post-exposure in proliferative tissues C_LIO_LIMultiple tissues exhibited the canonical ENU mutation spectrum 7 d after exposure C_LIO_LIResults obtained with DuplexSeq were highly concordant between laboratories C_LIO_LIThe Rat-50 Mutagenesis Assay is promising for applications in genetic toxicology C_LI

genomics↗

bioRxiv2023

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. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with >80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.

genomics↗

bioRxiv2023

Error-corrected Duplex Sequencing enables direct detection and quantification of mutations in human TK6 cells with remarkable inter-laboratory consistency

Error-corrected Duplex Sequencing (DuplexSeq) enables direct quantification of low-frequency mutations and offers tremendous potential for chemical mutagenicity assessment. We investigated the utility of DuplexSeq 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. In two independent experiments in two laboratories, TK6 cells were exposed to ENU (25-200 {micro}M) and DNA was sequenced 48, 72, and 96 h post-exposure. A DuplexSeq mutagenicity panel targeting twenty 2.4-kb regions distributed across the genome was used to sample diverse, genome-representative sequence contexts. A robust increase in MF that was unaffected by time was observed in both laboratories. Concentration-response in the MF from the two laboratories was strongly positively correlated (R2=0.95). C:G>T:A, T:A>C:G, T:A>A:T, and T:A>G:C mutations increased in consistent, concentration-dependent manners in both laboratories, with high proportions of C:G>T:A at all time points. The target sites responded similarly between the two laboratories and revealed a higher average MF in intergenic regions. These results, demonstrating remarkable reproducibility across time and laboratory for both MF and spectrum, support the high value of DuplexSeq for characterizing chemical mutagenicity in both research and regulatory evaluation.

genomics↗

bioRxiv2022

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 strategies for comparable detection of rare structural variant (SV) junctions, where different error mechanisms must be addressed. Working from cell 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. DuplexSeq could not address frequent intermolecular ligation artifacts that arise during Y-adapter addition prior to strand denaturation without requiring multiple source molecules. In contrast, tagmentation libraries coupled with data filtering based on strand family size greatly reduced both artifact classes and enabled efficient and specific detection of even single-molecule SV junctions. The throughput of SV capture sequencing (svCapture) and the high base-level accuracy of DuplexSeq provided detailed views of the microhomology profile and limited occurrence of de novo SNVs near the junctions of hundreds of sub-clonal and newly created SVs, suggesting end joining as a predominant formation mechanism. The open source svCapture pipeline enables rare SV detection as a routine addition to SNVs/indels in properly prepared capture sequencing libraries.

genomics↗

bioRxiv2020

Direct Quantification of in vivo Mutagenesis Using Duplex Sequencing

The ability to accurately measure mutations is critical for basic research and identification of 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 dont fully represent other species such as humans. Next generation sequencing (NGS) is a conceptually attractive alternative for mutation detection in the DNA of any organism, however, the limit of resolution for standard NGS is poor. Technical error rates (~1x10-3) of NGS obscure the true abundance of somatic mutations, which can exist at pernucleotide frequencies [≤]1x10-7. Using Duplex Sequencing, an extremely accurate error-corrected NGS (ecNGS) technology, we were able to detect mutations induced by 3 carcinogens in 5 tissues of 2 strains of mice within 31 days following exposure. We observed a strong correlation between mutation induction measured by Duplex Sequencing and the gold-standard transgenic rodent mutation assay. We identified exposure-specific mutation spectra of each compound through trinucleotide patterns of base substitution. We observed variation in mutation susceptibility by genomic region, as well as by DNA strand. We also identified the primordial signs of carcinogenesis in a cancer-predisposed strain of mice, as evidenced by clonal expansions of cells carrying an activated oncogene, less than a month after carcinogen exposure. These findings demonstrate that ecNGS is a powerful method for sensitively detecting and characterizing mutagenesis and the early clonal evolutionary hallmarks of carcinogenesis. Duplex Sequencing can be broadly applied to chemical safety testing, basic mutational research, and related clinical uses. SIGNIFICANCE STATEMENTError-corrected next generation sequencing (ecNGS) can be used to rapidly detect and quantify the in vivo mutagenic impact of environmental exposures or endogenous processes in any tissue, from any species, at any genomic location. The greater speed, higher scalability, richer data outputs, as well as cross-species and cross-locus applicability of ecNGS compared to existing methods make it a powerful new tool for mutational research, regulatory safety testing, and emerging clinical applications.

genomics↗

bioRxiv2019

Unexpectedly High Subclonal Mutational Diversity in Human Colorectal Cancer and Its Significance

Human colorectal cancers (CRC) contain both clonal and subclonal mutations. Clonal mutations are positively selected, present in most cells and drive malignant progression. Subclonal mutations are randomly dispersed throughout the genome; they provide a vast reservoir of mutant cells that can expand, repopulate the tumor and result in the rapid emergence of resistance, as well as being a major contributor to tumor heterogeneity. Here, we apply Duplex Sequencing (DS) methodology to quantify subclonal mutations in CRC tumor with unprecedented depth (104) and accuracy (<10-7). We measured mutation frequencies in genes encoding replicative DNA polymerases and in genes frequently mutated in CRC, and found an unexpectedly high effective mutation rate, 7.1 x 10-7. The curve of subclonal mutation accumulation as a function of sequencing depth, using DNA obtained from five different tumors, is in accord with a neutral model of tumor evolution. We present a new theoretical approach to model neutral evolution independent of the infinite sites assumption (which states that a particular mutation arises only in one tumor cell at any given time). Our analysis indicates that the infinite sites assumption is not applicable once the number of tumor cells exceeds the reciprocal of the mutation rate, a circumstance relevant to even the smallest clinically diagnosable tumor. Our methods allow accurate estimation of the total mutation burden in clinical cancers. Our results indicate that no DNA locus is wild type in every malignant cell within a tumor at the time of diagnosis (probability of all cells wild type = 10-308).\n\nSignificance StatementCancers evolve many mutations. Clonal mutations are selected early. Subsequent evolution occurs in a branching fashion, possibly without selection (\"neutral evolution\"). Rarer mutations occur later on smaller branches of the evolutionary tree. Using a DNA sequencing method, duplex sequencing, with unprecedented accuracy and sensitivity, we quantified very rare unique subclonal mutations in diagnostic specimens from five human colorectal cancers. Rarer subclones probe later evolutionary timepoints than previously possible. We confirm neutral evolution at later times and find many more subclonal mutations than expected. A novel theoretical method allowed us to extrapolate further forward in time to diagnosis. At diagnosis, every base in DNA is mutated in at least one cancer cell. In particular any therapy resistance mutation would be present.

cancer biology↗

bioRxiv2018

Ultra-sensitive 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. These mutations increase with age and share the selection traits of clonal TP53 mutations commonly found in human tumors. We show that low frequency TP53 mutations exist in multiple healthy tissues, from newborn to centenarian, and progressively increase in abundance and pathogenicity with older age across tissue types. Our results illustrate that subclonal cancer evolutionary processes are a ubiquitous part of normal human aging and great care must be taken to distinguish tumor-derived, from age-associated mutations in high sensitivity clinical cancer diagnostics.

cancer biology↗

bioRxiv2017

CRISPR-DS: An efficient, low DNA input method for ultra-accurate sequencing

Current next-generation sequencing techniques suffer from inefficient target enrichment and frequent errors. To address these issues, we have developed a targeted genome fragmentation approach based on CRISPR/Cas9 digestion. By designing all fragments to similar lengths, regions of interest can be size-selected prior to library preparation, increasing hybridization capture efficiency. Additionally, homogenous length fragments reduce PCR bias and maximize read usability. We combine this novel target enrichment approach with ultra-accurate Duplex Sequencing. The result, termed CRISPR-DS, is a robust targeted sequencing technique that overcomes the inherent challenges of small target enrichment and enables the detection of ultra-low frequency mutations with small DNA inputs.

genomics↗