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Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors
Published on: September 20, 2016
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Coverage analysis in a targeted amplicon-based next-generation sequencing panel for myeloid neoplasms
Benedict Yan1, Yongli Hu2, Christopher Ng1
1Department of Laboratory Medicine, Molecular Diagnosis Centre, National University Health System, Singapore, Singapore.
Journal of Clinical Pathology
|February 21, 2016
Summary
This study identifies problematic DNA regions in a myeloid next-generation sequencing panel, improving gene sequencing depth for better myeloid neoplasm diagnostics. A method was developed to increase read depth for the CEBPA gene.
Area of Science:
- Molecular Biology
- Genomics
- Clinical Diagnostics
Background:
- Next-generation sequencing (NGS) panels are vital for clinical diagnostics, but PCR amplicon-based methods face amplification bias.
- This bias can lead to uneven read depth, potentially affecting diagnostic accuracy for myeloid neoplasms.
Purpose of the Study:
- To characterize low-performing amplicons in the TruSight Myeloid Sequencing Panel.
- To investigate strategies for improving sequencing depth, specifically for the CEBPA gene.
Main Methods:
- Targeted NGS was performed on 158 acute myeloid leukemia samples, analyzing 568 amplicons for read depth.
- Correlation analysis was conducted between amplicon read depth and template GC content.
- A parallel library preparation workflow (Nextera XT) was used to address low read depth in CEBPA.
Main Results:
- Sixteen lower-performance amplicons impacting nine genes, including CEBPA, were identified.
- A slight negative correlation was observed between amplicon read depth and template GC content.
- Combining TruSight Myeloid and Nextera XT libraries improved CEBPA minimum read depth significantly across samples.
Conclusions:
- Identifying low-performance amplicons is crucial for laboratories using this NGS panel.
- Combining different library preparation methods on the same flow cell is a viable strategy to enhance sequencing depth for specific genes like CEBPA.
- This approach offers a proof-of-concept for optimizing NGS panel performance in myeloid diagnostics.
