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Amplicon Sequencing using the Long-Read Sequencing Technologies
Published on: August 29, 2025
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Targeted long-read nanopore sequencing as a complementary approach for detecting STRC variants and distinguishing the
Hideaki Moteki1,2, Shin-Ya Nishio3, Shin-Ichi Usami3
1Department of Hearing Implant Sciences, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, 390-8621, Nagano, Japan. moteki@shinshu-u.ac.jp.
Scientific Reports
|December 22, 2025
Summary
Long-read sequencing precisely detects hearing loss variants in the complex STRC gene region. This method overcomes challenges posed by the STRC pseudogene, aiding in diagnosing sensorineural hearing loss (SNHL).
Area of Science:
- Genetics
- Genomic Sequencing
- Hearing Loss Research
Background:
- The Stereocilin (STRC) gene is crucial for mild-to-moderate sensorineural hearing loss (SNHL), especially with deletions.
- The highly homologous STRC pseudogene (STRCP1) complicates the detection of single nucleotide variants (SNVs) and indels in the STRC gene.
- Conventional sequencing methods struggle to accurately analyze variants in the STRC gene region due to pseudogene interference.
Purpose of the Study:
- To evaluate targeted long-read sequencing using the nanopore MinION platform for detecting SNVs and indels in the STRC gene.
- To assess the efficacy of combining long-read sequencing with long-range PCR for enhanced variant detection in complex genomic regions.
- To identify novel pathogenic variants associated with SNHL in individuals with heterozygous STRC deletions.
Main Methods:
- Conducted targeted long-read sequencing on 149 DNA samples from individuals with SNHL phenotypes and heterozygous STRC deletions.
- Employed long-range polymerase chain reaction (PCR) enrichment coupled with nanopore MinION sequencing.
- Analyzed sequencing data for SNVs and small indels within the STRC gene region, accounting for pseudogene interference.
Main Results:
- Sequencing yielded a median of 4,088 reads with a median depth of 2,780, and an average read length of 20.76 kbp.
- Identified 43 individuals with SNVs or small indels, including 13 previously reported and 14 novel variants.
- Demonstrated precise variant detection in the complex STRC locus, overcoming pseudogene interference.
Conclusions:
- Targeted long-read sequencing is a valuable tool for precisely detecting pathogenic variants in complex genomic regions like STRC.
- This approach complements short-read next-generation sequencing (NGS) for resolving challenging cases of unresolved SNHL.
- The combination of long-read sequencing and long-range PCR significantly improves the resolution and accuracy of SNV detection in the STRC gene.
