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

HiFi long-read genome sequencing (PacBio ~30×) in real-world clinical laboratory conditions

HiFi long-read genome sequencing (PacBio, ~30×) was applied to 191 probands enriched for difficult-to-detect pathogenic variants across a real clinical laboratory. The automated pipeline detected 479 of 481 expected variants (99.6%), including repeat expansions, deep intronic variants, complex SVs, and mosaic variants — even from suboptimal samples such as buccal swabs and low-molecular-weight DNA. Only two mosaic variants at low allele fractions (trisomy 18 at 23% and a deletion at 13%) were missed.

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This study provides one of the strongest clinical-grade validations of HiFi long-read WGS in a diagnostic laboratory setting. The 99.6% detection rate across all difficult-to-detect variant classes — on imperfect real-world samples — is a critical milestone for moving beyond the sequential multi-assay paradigm. The residual blind spot for low-level mosaicism (<23%) is shared with current technologies. Single first-tier long-read WGS now appears within practical reach.

Clinical impact : 3/3 · Evidence strength : 3/3 · Novelty : 1/2 · Sample size : 1/1 · Journal quality : 1/1 → Total : 9/10