[Comparison of different massive parallel sequencing platforms for mutation profiling in formalin-fixed and...

Area of Science:

• Molecular Biology
• Genomics
• Oncology

Background:

• Next-generation sequencing (NGS) is crucial for molecular characterization of tumors.
• Formalin-fixed and paraffin-embedded (FFPE) samples present unique challenges for NGS due to potential DNA degradation.
• Comparing different NGS platforms is essential for optimizing clinical diagnostics.

Purpose of the Study:

• To evaluate and compare the performance of Miseq and Ion Torrent PGM platforms for NGS.
• To assess the efficacy of these platforms in analyzing DNA from FFPE cancer samples.
• To determine the optimal NGS platform for reliable mutation detection in FFPE tissues.

Main Methods:

• Retrospective analysis of 204 FFPE cancer samples (100 non-small cell lung, 104 colorectal).
• DNA extraction and library preparation using identical kits and protocols for both platforms.
• Sequencing performed on Miseq and Ion Torrent PGM platforms, followed by validation of discordant mutations using ARMS-PCR and Sanger sequencing.

Main Results:

• Miseq platform generated significantly higher numbers of reads, superior alignment rates, and greater median sequence depth compared to PGM.
• A high concordance (93.6%) of detected mutations was observed between the two platforms.
• Miseq demonstrated higher reliability for detecting low-frequency mutations, with discordant results often attributed to low mutation frequency or sample age.

Conclusions:

• Miseq platform exhibits superior sequencing quality and reliability for analyzing FFPE samples over Ion Torrent PGM.
• The choice of NGS platform should consider sample characteristics, throughput needs, and desired reliability for molecular tumor profiling.
• Miseq is recommended for more dependable molecular characterization of FFPE cancer specimens.