Assessing Mutagenicity in Cells or Tissues
Generate high-confidence mutation data of your substances
SMM-Seq is a highly sensitive error-corrected next-generation sequencing (ecNGS) approach for measuring exposure-induced DNA mutations. ecNGS can be applied in vitro or in vivo, using tissues repurposed from repeat-dose general toxicity studies.
SMM-seq provides accurate and detailed information about mutation frequency and the spectrum of induced mutations.
Whether you are investigating a positive Ames result, looking for a human-relevant in vitro mutation assay, or studying mutagenesis in vivo, SMM-seq can help you obtain reliable mutation data.
Benefits
- Quantifies mutation frequency in cells and tissues
- Provides mutation spectra information
- Genome-wide and untargeted approach
- Unbiased and highly sensitive
- Represents a potential alternative to the transgenic rodent assays
- Technology How does it work?
- Applications Is this for me?
- Validation Can I trust the data?
- Resources
SMM-seq is an error-corrected next-generation sequencing (ecNGS) method that combines rolling circle amplification (RCA) with next-generation sequencing to detect rare DNA mutations with exceptionally high accuracy. As SMM-seq uses a genome-wide untargeted approach, mutations can be assessed across the genome rather than being limited to predefined target regions.
Steps in rolling circle amplification:
- Total genomic DNA from cells or tissues is fragmented
- Hairpin-shaped adapters are attached to both ends of the DNA fragment, forming a circular molecule
- The circular DNA is copied multiple times using RCA, generating many independent copies of each strand in the original double-stranded DNA molecule
- After DNA quantification, samples are pooled and analyzed using Illumina sequencing
As all copies originate from the same parent molecule, you can:
- Identify true mutations that are present in every copy
- Filter out random alterations that appear inconsistently due to DNA amplification and sequencing errors.
Do you want to get more detailed information?
In vitro mutagenicity assay
If your substance produces a positive Ames test result, SMM-seq can provide additional data to help determine whether further development is warranted.
- More informative than Hprt and Mouse Lymphoma Assay (MLA)
- Gives the same mutation frequency and spectrum endpoints used in in vivo ecNGS studies
- Provides deeper insights about the mutagenic mode of action (MoA)
SMM-seq can also be applied if:
- Your test article is incompatible with a bacterial mutation assay (e.g. antibiotics)
- You want to investigate mutagenesis in more physiological 2D and 3D cell models
In vivo mutagenicity study
Reducing animal testing is a shared goal in the toxicology community. Exploring alternatives to transgenic rodent mutagenicity assays is possible with SMM-seq.
- Leverage tissue samples already collected during your 28-, 90-day repeat dose animal study
- Reduce the need to run a dedicated transgenic rodent gene mutation study.
- Reduce costs
In vitro validation of SMM-seq with mouse embryonic stem cells
As part of the validation, 22 known mutagenic and non-mutagenic compounds were tested, and the results were compared to standard mutagenicity assays (Ames and TGR).
Mouse embryonic stem cells were exposed to different concentrations of each compound for 4 hours. Five days post-exposure, cells were prepared for DNA extraction and sequencing.
Mutation frequency and mutation spectra information showed the mutagenicity and mode of action expected for each compound.
As an example, the pattern of point mutations induced by ENU was dominated by A:T –> T:A transversions and A:T –> G:C transitions, consistent with its known mechanism as an SN1 alkylating agent and its canonical mutation spectrum. A different example is potassium bromate, a known oxidative stress activator, which shows a high percentage of C > A mutations, corresponding to its known mode of action (MoA).
Download the flyer to see the full table of results for the 22 compounds and their comparison to the standard mutagenicity assays.
Steps to get your SMM-seq mutagenicity data
Toxys offers SMM-seq through a partnership with Mutagentech.
We handle sample preparation and coordinate advanced sequencing with our partner. Alternatively, you can send us your test articles, and we can perform the in vitro exposure.
The Toxys expert team then prepares an informative report that interprets the data and meets with you to provide clear insights.
Looking for a mammalian in vitro assay that predicts mutagenicity and genotoxicity?
Discover MutaTracker: A combination of ToxTracker with SMM-seq covering the main genotoxicity endpoints and detecting single-point mutations in one assay.
Contact our team
Inger Brandsma, Scientific Director at Toxys
