Parallel Analysis of Repeat Expansions: An Updated Clinical Nanopore Cas9-Targeted Sequencing Workflow for Nanopore R10 Flow Cells.

Cas9-targeted nanopore sequencing on R10 flow cells, with base-called reads analysed through the Epi2Me wf-human-variation workflow and the Straglr tool

Hereditary ataxias caused by short tandem repeat expansions are difficult to diagnose by PCR and Southern blot, which handle complex expansions poorly and cannot assess repeat interruptions or methylation. The authors present an updated clinical Cas9-targeted nanopore sequencing workflow, now compatible with Oxford Nanopore Technologies R10 flow cells. It incorporates the Epi2Me wf-human-variation workflow and the Straglr tool applied to base-called reads, ensuring compatibility with past, current and future sequencing chemistries. The number of genes analysed rises from 10 to 27, with new panels for ataxia, myopathy, neurodegeneration and amyotrophic lateral sclerosis. Validated on Coriell reference and clinical samples, the workflow was also tested with multiplexing, Flongle flow cells and single-gene targeting: only single-gene targeting proved compatible and reliable.

Synthesis written by Geno'X. For the full original abstract, please refer to the source publication.

The value lies less in the gene count than in what targeted nanopore delivers and PCR does not: repeat structure, interruptions and methylation — three elements that change the interpretation of an intermediate allele or an atypical expansion. The negative result on multiplexing and Flongle flow cells is the most useful practical finding: it closes off the most tempting route to lower per-patient cost. The abstract reports no quantitative sensitivity or specificity, leaving local validation entirely to the adopting laboratory.

Analysis by Dr Thibaut Benquey