Parallel Analysis of Repeat Expansions

Journal of Molecular Diagnostics

Abstract

Hereditary ataxias, caused by expansions of short tandem repeats, are difficult to diagnose using traditional PCR and Southern blot methods, which struggle to detect complex repeat expansions and cannot assess repeat interruptions or methylation. An updated Clinical Nanopore Cas9-Targeted Sequencing workflow is presented for analyzing repeat expansions, now compatible with the Oxford Nanopore Technologies R10 flow cell. The workflow incorporates the Oxford Nanopore Technologies wf-human-variation Epi2Me workflow, including the Straglr tool to analyze base-called reads, ensuring compatibility with past, current, and future sequencing chemistries. It expands the number of genes analyzed from 10 to 27 and introduces new gene panels for ataxia, myopathy, neurodegeneration, and amyotrophic lateral sclerosis/motor neuron disease. Validated with Coriell reference and clinical samples, this method improves the analysis of pathogenic repeat expansions, providing deeper insights into repeat structures while addressing the limitations of traditional approaches. In this work, the use of multiplexing, Flongle flow cells, and single-gene targeting were explored as alternatives to panel-based approaches in the Clinical Nanopore Cas9-Targeted Sequencing workflow, finding that only single-gene targeting provides compatibility and reliable performance.

Authors 22

1. Veronika Scholz

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

2. Veronika Schönrock

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

3. Hannes Erdmann

   Medical Genetics Center · Ludwig-Maximilians-Universität München

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany; Friedrich-Baur-Institute, Department of Neurology, Klinikum der Universität, Ludwig-Maximilians-Universität, Munich, Germany

4. Vitus Prokosch

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

5. Maria Schoedel

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

6. Manuela Almus

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

7. Mayra Sauer

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

8. Veronika Mayer

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

9. Eva Breithausen

   Medical Genetics Center

   Affiliation as printed

   MGZ-Medical Genetics Center, Munich, Germany

10. Inga Van Buren

    Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany

11. Christine Dineiger

    Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany

12. M Golibrzuch

    Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany

13. Minerva Montero-Hernández

    Medical Genetics Center · University of Castilla-La Mancha

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Laboratorio de Genética Médica, Instituto de Biomedicina, Facultad de Medicina de Albacete, Universidad de Castilla-La Mancha, Spain

14. Annette Lischka Aachen Center for Human Genetics and Genomic Medicine Faculty of Medicine

    RWTH Aachen University

    Affiliation as printed

    Center for Human Genetics and Genomic Medicine, Faculty of Medicine, RWTH Aachen University, Aachen, Germany

15. Katja Eggermann Aachen Center for Human Genetics and Genomic Medicine Faculty of Medicine

    RWTH Aachen University

    Affiliation as printed

    Center for Human Genetics and Genomic Medicine, Faculty of Medicine, RWTH Aachen University, Aachen, Germany

16. Caroline Heintz

    Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany

17. Ariane Hallermayr

    LMU Klinikum · Medical Genetics Center · Ludwig-Maximilians-Universität München

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Medizinische Klinik und Poliklinik IV, Klinikum der Universität München Campus Innenstadt, Munich, Germany

18. Teresa Neuhann

    Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany

19. Elke Holinski‐Feder

    LMU Klinikum · Medical Genetics Center · Ludwig-Maximilians-Universität München

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Medizinische Klinik und Poliklinik IV, Klinikum der Universität München Campus Innenstadt, Munich, Germany

20. Angela Abicht

    Medical Genetics Center · Ludwig-Maximilians-Universität München

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Friedrich-Baur-Institute, Department of Neurology, Klinikum der Universität, Ludwig-Maximilians-Universität, Munich, Germany

21. Anna Benet‐Pagès

    Helmholtz Munich · Medical Genetics Center

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Institute of Neurogenomics, Helmholtz Zentrum München GmbH-German Research Center for Environmental Health, Munich, Germany

22. Morghan C. Lucas corresponding

    LMU Klinikum · Medical Genetics Center · Ludwig-Maximilians-Universität München

    Affiliation as printed

    MGZ-Medical Genetics Center, Munich, Germany; Friedrich-Baur-Institute, Department of Neurology, Klinikum der Universität, Ludwig-Maximilians-Universität, Munich, Germany; Medizinische Klinik und Poliklinik IV, Klinikum der Universität München Campus Innenstadt, Munich, Germany. Electronic address: morghan.lucas@mgz-muenchen.de