Sensitivity of HiFi long-read genome sequencing for difficult-to-detect pathogenic variants when applied to real-world clinical laboratory samples.

Leveraging new sequencing and omic technologies to enhance the detection of pathogenic variants in known disease genes is a key step toward increasing the likelihood of a precise genetic diagnosis for affected individuals. Short-read sequencing is widely used in clinical laboratories for multi-gene panels and exome and genome sequencing, but this technology has inherent limitations in detecting certain classes of genetic variation. As a result, diagnostic laboratories continue to offer complementary assays, often used sequentially, reducing efficiency and speed in providing a diagnosis. We applied PacBio long-read genome sequencing (HiFi) to samples from 191 probands previously tested with short-read sequencing alone and/or other diagnostic technologies and enriched for pathogenic variants difficult to detect (VDDs). HiFi's pipeline automatically detected 479 of 481 (99.6%) disease-causing variants, many of which were called in samples not optimized for long-read genome sequencing (such as buccal samples or low-molecular-weight DNA). The two variants not automatically detected were a mosaic trisomy 18 (23% mosaicism) and a 5,594-bp mosaic deletion (13% mosaicism). However, other mosaic variants were detected, indicating that HiFi at ∼30× genome coverage is sensitive to the degree of mosaicism. Of 481 variants, 49 were suspected based on the clinical report but not confirmed molecularly prior to HiFi. Our findings demonstrate that HiFi sequencing detects a wide range of VDDs in real-world clinical laboratory samples, highlighting a key advantage of HiFi as a potential first-tier test over the myriad of complementary technologies currently used to detect VDDs.