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Updated: May 10, 2026
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Rare Event Detection Using Error-corrected DNA and RNA Sequencing
Published on: August 3, 2018
Routine performance and errors of 454 HLA exon sequencing in diagnostics
Norbert Niklas1, Johannes Pröll, Martin Danzer
1Red Cross Transfusion Service for Upper Austria, Krankenhausstraße 7, 4017 Linz, Austria. norbert.niklas@o.roteskreuz.at
BMC Bioinformatics
|June 5, 2013
Summary
Next-generation sequencing (NGS) error rates are now low enough for diagnostic applications. This study details error assessment in human leukocyte antigen (HLA) exon sequencing using the 454 GS Junior system.
Area of Science:
- Genomics
- Molecular Biology
- Bioinformatics
Background:
- Next-generation sequencing (NGS) has revolutionized genomics, with increasing applications across research fields.
- Accurate determination of sequencing errors is crucial for advancing NGS beyond research into clinical diagnostics.
- This study focuses on the 454 GS Junior system for human leukocyte antigen (HLA) exon sequencing.
Purpose of the Study:
- To assess the performance and error profile of the 454 GS Junior system for diagnostic HLA exon sequencing.
- To characterize sequencing errors and variants in a validated diagnostic assay.
- To evaluate the feasibility of NGS for diagnostic applications based on error rates.
Main Methods:
- Utilized multiple runs of the 454 GS Junior system with an established diagnostic assay for HLA exon sequencing.
- Sequenced 60 HLA loci per run.
- Extracted, evaluated, and characterized errors and variants based on sequence adjacencies.
Main Results:
- An overall error rate of 0.18% was determined across 118,484,408 bases.
- 31.3% of reads contained errors, with deletions constituting 50% of all errors.
- Errors were not uniformly distributed, tending to occur at sequence ends and specific internal positions, often correlating with lower quality scores.
Conclusions:
- This is the first error assessment of a human NGS diagnostic assay using an amplicon sequencing approach.
- Sequence quality and error rates have improved, reaching a level suitable for diagnostic applications.
- The data confirm the relationship between homopolymers and errors, and suggest that while error correction tools are not essential, they can enhance efficiency.
