TRACE: Open-source software for quantifying somatic variation of tandem repeats by capillary electrophoresis

Abstract

Expanded short tandem DNA repeats are implicated in over 60 human disorders. In many, somatic instability (SI) of the repeat plays a critical role in disease pathogenesis. For example, SI in vulnerable neurons is a key driver of clinical symptoms in Huntington's disease. Quantifying SI has traditionally relied on PCR followed by capillary electrophoresis, with metrics describing the shape of repeat size distributions, such as the expansion index. However, current tools often require costly proprietary software, are time-consuming, and rely on custom pipelines that vary between labs. To address these challenges, we developed Tandem Repeats Analysis by Capillary Electrophoresis (TRACE), an open-source software that processes fragment analysis data end-to-end, from raw files to SI metrics. Additionally, we created an associated web app TRACE-shiny (https://traceshiny.mgh.harvard.edu), for interactive usage. Instability metrics from TRACE benchmarked against published datasets confirm its utility for studying genetic and pharmacological modifiers of SI. TRACE eliminates the need for proprietary software or custom pipelines, making advanced tools for analysis of somatic repeat expansion widely accessible.

Keywords

cag repeat instability repeat expansion dynamics and biochemistry repeat instability cag repeat expansions genetics

Get full access to this article

View all access options for this article.

References

Depienne

Mandel

J-L

. 30 Years of repeat expansion disorders: what have we learned and what are the remaining challenges? Am J Hum Genet 2021; 108: 764–785.

Chen

, et al. Repeat expansion disorders. Pract Neurol 2025; 25: 204–216.

Genetic Modifiers of Huntington’s Disease (GeM-HD) Consortium Genetic modifiers of somatic expansion and clinical phenotypes in Huntington's disease highlight shared and tissue-specific effects. Nat Genet 2025; 57: 1426–1436.

Mätlik

, et al. Cell-type-specific CAG repeat expansions and toxicity of mutant huntingtin in human striatum and cerebellum. Nat Genet 2024; 56: 383–394.

Swami

, et al. Somatic expansion of the Huntington's disease CAG repeat in the brain is associated with an earlier age of disease onset. Hum Mol Genet 2009; 18: 3039–3047.

Lee

, et al. A novel approach to investigate tissue-specific trinucleotide repeat instability. BMC Syst Biol 2010; 4: 29.

Mouro Pinto

, et al. In vivo CRISPR–Cas9 genome editing in mice identifies genetic modifiers of somatic CAG repeat instability in Huntington’s disease. Nat Genet 2025; 57: 314–322.

Mouro Pinto

, et al. Patterns of CAG repeat instability in the central nervous system and periphery in Huntington's disease and in spinocerebellar ataxia type 1. Hum Mol Genet 2020; 29: 2551–2567.

Chastain

2nd , et al. Anomalous rapid electrophoretic mobility of DNA containing triplet repeats associated with human disease genes. Biochemistry 1995; 34: 16125–16131.

10.

McAllister

, et al. Exome sequencing of individuals with Huntington's disease implicates FAN1 nuclease activity in slowing CAG expansion and disease onset. Nat Neurosci 2022; 25: 446–457.

11.

Covarrubias-Pazaran

, et al. Fragman: an R package for fragment analysis. BMC Genet 2016; 17: 62.

12.

McLean

, et al. Splice modulators target PMS1 to reduce somatic expansion of the Huntington’s disease-associated CAG repeat. Nat Commun 2024; 15: 3182.

13.

Charif

Lobry

. Seqinr 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis. In: Bastolla

, et al. (eds) Structural approaches to sequence evolution: molecules, networks, populations. Berlin, Heidelberg: Springer Berlin Heidelberg, 2007, pp.207–232.

14.

Southern

. Measurement of DNA length by gel electrophoresis. Anal Biochem 1979; 100: 319–323.

15.

Wood

. Fast stable restricted Maximum likelihood and marginal likelihood estimation of semiparametric generalized linear models. J R Stat Soc Ser B Stat Methodol 2010; 73: 3–36.

16.

Wheeler

, et al. Length-Dependent gametic CAG repeat instability in the Huntington's disease knock-in mouse. Hum Mol Genet 1999; 8: 115–122.

17.

Menalled

Chesselet

M-F

. Mouse models of Huntington's disease. Trends Pharmacol Sci 2002; 23: 32–39.

18.

Ferguson

, et al. Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease. Am J Hum Genet 2024; 111: 1165–1183.

19.

Bates

, et al. Fitting linear mixed-effects models using lme4. J Stat Softw 2015; 67: 1–48.

20.

Genetic Modifiers of Huntington’s Disease

(GeM-HD) Consortium

. CAG Repeat not polyglutamine length determines timing of Huntington's disease onset. Cell 2019; 178: 887–900.e14.

21.

Mejia Maza

, et al. MSH3 Is a genetic modifier of somatic repeat instability in X-linked dystonia parkinsonism. Am J Hum Genet 2026; 113: 83–99.

22.

Shalabh. Interactive web-based data visualization with R, plotly, and shiny. J R Stat Soc Ser A Stat Soc 2021; 184: 1150–1150.

23.

Ciosi

, et al. Approaches to sequence the HTT CAG repeat expansion and quantify repeat length variation. J Huntingtons Dis 2021; 10: 53–74.

24.

Maestri

, et al. Navigating triplet repeats sequencing: concepts, methodological challenges and perspective for Huntington’s disease. Nucleic Acids Res 2025; 53(1): gkae1155.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.62 MB

0.00 MB