The Utility of Ultra-Deep RNA Sequencing in Mendelian Disorder Diagnostics

Authors

Sen Zhao
Jefferson C Sinson
Shenglan Li
Jill A Rosenfeld
Gladys Zapata
Kristina Macakova
Mezthly Pena
Becky Maywald
Kim C Worley
Lindsay C Burrage
Monika Weisz-Hubshman
Shamika Ketkar
William Craigen
Lisa Emrick
Undiagnosed Diseases Network
Tyson Clark
Gila Yanai Lithwick
Zohar Shipony
Christine Eng
Brendan Lee
Pengfei Liu

Language

English

Publication Date

11-6-2025

Journal

American Journal of Human Genetics

DOI

10.1016/j.ajhg.2025.09.013

PMID

41075783

PMCID

PMC12777520

PubMedCentral® Posted Date

1-8-2026

PubMedCentral® Full Text Version

Author MSS

Abstract

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.

Keywords

Humans, Genetic Diseases, Inborn, Sequence Analysis, RNA, High-Throughput Nucleotide Sequencing, RNA Splicing

Published Open-Access

yes

Recommended Citation

Zhao, Sen; Sinson, Jefferson C; Li, Shenglan; et al., "The Utility of Ultra-Deep RNA Sequencing in Mendelian Disorder Diagnostics" (2025). Faculty, Staff and Students Publications. 6250.
https://digitalcommons.library.tmc.edu/baylor_docs/6250