The utility of ultra-deep RNA sequencing in Mendelian disorder diagnostics
Open accessAbstract
RNA sequencing (RNA-seq) has emerged as a powerful tool for resolving variants of uncertain significance (VUSs), particularly those affecting gene expression and splicing. However, most reference datasets and diagnostic protocols employ relatively modest sequencing depths (∼50-150 million reads), which may fail to detect low-abundance transcripts and rare splicing events critical for accurate diagnosis. We evaluated the diagnostic and translational utility of ultra-high-depth (up to ∼1 billion unique reads) RNA-seq in four clinically accessible tissues using the Ultima sequencing platform. After validating the performance of Ultima RNA-seq, we investigated how increasing sequencing depth affects gene and isoform detection, splicing variant discovery, and clinical interpretation of VUSs. Deep RNA-seq substantially improved sensitivity for detecting lowly expressed genes and isoforms, achieving near saturation for detection at 1 billion reads. In two probands with VUSs, pathogenic splicing abnormalities were undetectable at 50 million reads but emerged at 200 million reads, becoming even more pronounced at 1 billion reads. Using deep RNA-seq data, we constructed a resource, MRSD-deep, to estimate the minimum required sequencing depth to achieve desired coverage thresholds. MRSD-deep provided gene- and junction-level guidelines, helping labs select appropriate coverage targets for specific applications. Leveraging deep RNA-seq data on fibroblasts, we also built an expanded splicing-variation reference that successfully identified low-abundance splicing events missed by standard-depth data. Our findings underscore the diagnostic and research benefits of deep RNA-seq for Mendelian disease investigations.
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The sequencing depth achieved ultra-deep RNA-Seq coverage sufficient for both RNA expression and differential alternative splicing analysis.
“This sequencing depth achieved ultra-deep RNA-Seq coverage sufficient for both RNA expression and differential alternative splicing analysis to aid in transcript isoform detection56.”
From EXOSC3 G191 Variants Trigger System-Wide Recalibration of RNA Processing Machinery (2026) · cites this paper as [56]
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- Authors
- Zhao, S., Sinson, J. C., Li, S., Rosenfeld, J. A., Zapata, G., Macakova, K., Pena, M., Maywald, B., Worley, K. C., Burrage, L. C., Weisz-Hubshman, M., Ketkar, S., Craigen, W., Emrick, L., Clark, T., Lithwick, G. Y., Shipony, Z., Eng, C., Lee, B., & Liu, P.
- Affiliations
- Baylor College of Medicine (US)
- Corresponding author
- Liu, Pengfei
- ORCID
- Venue
- The American Journal of Human Genetics
- Publisher
- Elsevier BV
- Published
- 2025-11
- Volume
- 112
- Issue
- 11
- Pages
- 2578-2590
- ISSN
- 0002-9297 (ISSN-L)
- PMC
- PMC12777520
- OpenAlex
- W4415045761
- Cited by
- 5
- References
- 38
- Citations by year
- 2026: 4 · 2025: 1
- FWCI
- 2.94
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- Locations
- The American Journal of Human Genetics (publishedVersion)
- medRxiv (acceptedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (submittedVersion)
- Digital Commons-TMC (Texas Medical Center) (submittedVersion)
- Funders
- National Human Genome Research Institute (R35HG011311)
- National Institutes of Health
- National Institute of Neurological Disorders and Stroke (U01HG007709, U01HG007942)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genomics and Rare Diseases
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Research and Splicing
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- monogenic disorders, variants of uncertain significance, splice variants, sequencing depth, low-abundance transcripts, isoform detection, fibroblast transcriptome
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