In Business Since 2004
The Pytheas Software Suite
Tools for Identifying RNA Modifications from MS2 Fragmentation Spectra
The Pytheas Software Suite
At Cassia, LLC, we continue to not only empower scientists with new cutting‑edge reagents, but also software tools to explore one of the most dynamic and influential areas of RNA research. The Pytheas2 software uses state‑of‑the‑art LC‑MS/MS data to accurately detect and identify nucleoside modifications in mRNA, rRNA, tRNA, and custom RNA samples. Our system is adaptive and can match every MS2 spectrum against the fragments your RNA of interest can produce, scoring each match, and tests them against decoy sequences, so every result comes with a false discovery rate. It is the engine behind Cassia’s RNA Mapping Service, offering comprehensive qualitative and quantitative insights into RNA modification landscapes. Pytheas2 represents a comprehensive redesign of Pytheas1, an open-source software package originally published in 2022 (D’Ascenzo et al., Nature Communications). Rebuilt from the ground up, this latest iteration introduces a modern interface, expanded functionality, and performance enhancements that increase processing speeds by approximately 200-fold.
Three Ways to Use Pytheas
| Pytheas1 | Pytheas2-Lite | Pytheas2 (Through Cassia) | |
|---|---|---|---|
| What is it? | Original Open-Source Pytheas Refreshed Desktop GUI in 2026 |
Pytheas2 Rebuilt from Pytheas1 With New Web Browser GUI |
Full Pytheas2 Engine Tech Support and/or Service |
| Who it is for? | Anyone | Academic Labs | Companies and Academic Projects Needing Full Features & Services |
| What it Runs? | Digest & Match On Your Computer |
Digest & Match Through Your Browser |
Digest & Match; Quantitation via Isotope Labels; Tolerance Tuning from Your Data; Discovery of Unexpected Modifications |
| Cost? | Free (MIT License) | Free for Academic Research | Collaboration or Fee-For-Service |
| How to Start? | Install from PyPI | Request an Account Sign In Here |
Tell Us About Your Project |
What Pytheas2 Does
- Digest: Builds every fragment your nuclease can make from your sequences, with known modifications, partial modification, end chemistries and heavy-isotope labels.
- Match: Scores every candidate for every spectrum and keeps close calls visible, such as a modification that could sit on either of two neighbouring nucleotides.
- False Discovery Rate: Every target sequence has a decoy of the same composition and mass, and each run reports how many identifications pass at each FDR.
- Results You Can Check: Sortable tables of every candidate, annotated spectra, sequence maps of each RNA, and exports in the standard idXML and mzTab-NA formats.
- Modification Library: Each residue is cross-referenced to the MODOMICS database.
What Data Inputs You Need
- RNA Sequence: Your RNA sequence in FASTA file format.
- MS2 Spectra: Your MS2 spectra should be in .mgf file format. Pytheas2 contains an MS Data Prep guide showing how to convert instrument files using ProteoWizard MSConvert.
- Modifications: Either the specific modification you expect, or use the built-in library.
Getting Started
- Academic Labs: Request a free Pytheas2-Lite account. We routinely approve requests within two (2) business days and email your sign-in details. Please use your institutional email address.
- ASMS Workshop Participants: Use the workshop account given out in the session.
- Companies & Special Projects Needing the Full Pytheas2 Engine: Tell us about your project. We’ll reply to discuss a collaboration or fee-for-service analysis. Please use your company or institutional email address.
- Have an Account?: Sign In Here
- Questions: rna_ms@cassiallc.com
How to Cite Pytheas
- D’Ascenzo L, Popova AM, Abernathy S, Sheng K, Limbach PA, Williamson JR. (2022) Pytheas: A Software Package for the Automated Analysis of RNA Sequences and Modifications via Tandem Mass Spectrometry. Nature Communications 13, 2424-2436. (https://doi.org/10.1038/s41467-022-30057-5)
Products and Services are for Laboratory Research Applications Only — not for drug, diagnostic, household, or other purposes. Persons intending to use these products and services for other applications do so at their own responsibility and must comply with all appropriate regulations.