TwinStrand DuplexSeq measuring MRD in AML


TwinStrand now offers mutagenicity testing services

TwinStrand's Mutagenesis Service Lab partners with clients and CROs to run DuplexSeq mutagenicity studies on repurposed in vivo tissues​

DuplexSeq Assays identify and count the specific types of mutations in your samples, yielding both mutation frequencies and data informing the mutagenic mechanism at play. Run a DuplexSeq Mutagenesis assay on wild-type rodent tissues repurposed from 28-day repeat dose general toxicology studies.​

Integrates seamlessly into any in vivo study workflow​

You can run DuplexSeqTM Mutagenesis Assays on commonly used tissues from transgenic or wild-type rodent studies. TwinStrand does not run animal studies – all in vivo DuplexSeq mutagenicity studies use repurposed tissues generated by partner labs​.


Numerous in vivo DuplexSeq Mutagenesis studies have been published by our clients and collaborators. Here is a selection of publications from groups that are applying DuplexSeq Mutagenesis assays for in vivo testing, including bridging studies to the transgenic rodent and Pig-a assays.

Adopting duplex sequencing technology for genetic toxicity testing: proof-of-concept mutagenesis experiment with N-ethyl-N-nitrosourea (ENU)-exposed rats​

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. 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 DS-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 spec-trum. 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.


Stephanie L. Smith-Roe a, Cheryl A. Hobbs b, Victoria Hull b, J. Todd Auman b, Leslie Recio b 1, Michael A. Streicker b, Miriam V. Rivas b, Gabriel A. Pratt c 2, Fang Yin Lo c 3, Jacob E. Higgins c, Elizabeth K. Schmidt c, Lindsey N. Williams c 4, Daniela Nachmanson c, Charles C. Valentine III c 5, Jesse J. Salk c, Kristine L. Witt

Error-corrected next-generation sequencing to advance nonclinical genotoxicity and carcinogenicity testing

Nature Reviews Drug Discovery, vol. 22, no. 3, Mar. 2023, pp. 165–66.
Error-corrected next-generation sequencing to advance nonclinical genotoxicity and carcinogenicity testing

Error-corrected next-generation sequencing (ecNGS) is an emerging technology with the potential to revolutionize the field of genetic toxicology. Here, we present recommendations from an expert working group convened to discuss potential applications, advantages and challenges associated with implementing ecNGS in nonclinical safety studies.


Francesco Marchetti, Renato Cardoso, Connie L. Chen, George R. Douglas, Joanne Elloway, Patricia A. Escobar, Tod Harper Jr, Robert H. Heflich, Darren Kidd, Anthony M. Lynch, Meagan B. Myers, Barbara L. Parsons, Jesse J. Salk, Raja S. Settivari, Stephanie L. Smith-Roe, Kristine L. Witt, Carole Yauk, Robert R. Young, Shaofei Zhang & Sheroy Minocherhomji

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. DS revealed a linear dose-related induction of mutations across all targets with low intra-group variability. Heterochromatic and intergenic regions exhibited the highest mutation frequencies (MF). C:G > A:T transversions at CCA, CCC and GCC trinucleotides were enriched in BaP-exposed mice consistent with the known etiology of BaP mutagenesis. However, GC-content had no effect on mutation susceptibility. A positive correlation was observed between DS and the “gold-standard” transgenic rodent gene mutation assay. Overall, we demonstrate that DS is a promising approach to study in vivo mutagenesis and yields critical insight into the genomic features governing mutation susceptibility, spectrum, and variability across the genome.


Danielle P. M. LeBlanc, Matthew Meier, Fang Yin Lo, Elizabeth Schmidt, Charles Valentine III, Andrew Williams, Jesse J. Salk, Carole L. Yauk & Francesco Marchetti

Comparison of the transgenic rodent mutation assay, error corrected next generation duplex sequencing, and the alkaline comet assay to detect dose-related mutations following exposure to N-nitrosodiethylamine

N-Nitrosodiethylamine (NDEA), a well-studied N-nitrosamine, was tested in rats to compare the dose-response relationship of three genotoxicity endpoints. Mutant / mutation frequencies were determined using the transgenic rodent (TGR) gene mutation assay and error corrected next generation sequencing (ecNGS) (i.e., duplex sequencing (DS)), and genetic damage was detected by the alkaline comet assay. Big Blue® (cII Locus) animals (n = 6 per dose group) were administered doses of 0.001, 0.01, 0.1, 1, 3 mg/kg/day NDEA by oral gavage. Samples were collected for cII mutation and DS analyses following 28-days of exposure and 3 days recovery. In a separate study, male Sprague-Dawley (SD) rats (n = 6 per dose group) were administered the same doses by oral gavage for two consecutive days and then samples collected for the alkaline comet assay. A dose-related increase in mutant / mutation frequencies of the liver but not duodenum was observed using the TGR assay and DS with DS resulting in a slightly more sensitive response, with a lower benchmark dose (BMD). In addition, a dose-related increase in percent tail DNA was observed in the liver using the alkaline comet assay. Therefore, DS and comet assays showed good utility for hazard identification and dose-response analysis of a representative N-nitrosamine comparable to the TGR gene mutation assay.


Bercu J., Zhang, S., Sobo, Z., Escobar, P., Van, P., Schuler, M.

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