Yield of Long-Read Genome Sequencing for Rare Disease Diagnosis in Short-Read Genome Negative Cases

Long-read genome sequencing paired with prior short-read data in the same cases, complemented by RNA-seq and CpG methylation profiling

One hundred forty-four cases previously analysed by short-read genome sequencing were re-sequenced with long-read genome sequencing; 107 of them remained undiagnosed. Thirteen new molecular diagnoses were made, five of which could not have been detected by short-read sequencing, an incremental diagnostic yield of 4.7%. These five diagnoses relied on long-read-specific capabilities: variants in short-read dark regions, structural variants and tandem repeat expansions, and de novo variant identification using only one biological parent. In two of the five cases, confirmation also required demonstration of aberrant transcript production by RNA-seq, and in another, long-read CpG methylation profiling supported the diagnosis through abnormal methylation at episignature-associated CpGs.

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

The number to remember is 4.7% in cases already negative on short-read genome sequencing: small in absolute terms, but these are diagnostic dead ends, and long-read delivers structural variants, repeat expansions, dark regions and methylation from a single assay. The limits are cost and the fact that three of the five cases required RNA-seq or methylation profiling to conclude: long-read alone does not always settle the case. A non-peer-reviewed preprint on a single-centre series of 144 cases — the figure needs confirmation in a larger cohort.

Analysis by Dr Thibaut Benquey