Benchmarking and optimization of a high-throughput sequencing based method for transgene sequence variant analysis in...

Area of Science:

• Biotechnology
• Molecular Biology
• Genomics

Background:

• High-Throughput Sequencing (HTS) is crucial for detecting mutations in biotherapeutic transgene products during cell line development.
• Previous work established an amplicon sequencing method for rapid mutation detection to aid cell line clone selection.
• Assay improvements were driven by practical experience across various cell line development programs.

Purpose of the Study:

• To report on improvements to an existing HTS-based mutation detection assay.
• To comprehensively benchmark the enhanced assay's performance and robustness.
• To identify optimal sample and library preparation protocols for minimizing errors and false positives.

Main Methods:

• Developed and refined an amplicon sequencing assay targeting transgene RNA for mutation detection.
• Benchmarked assay performance using spike-in mutations ranging from 60% down to 0.3% frequency.
• Evaluated the impact of 16 different sample and HTS library preparation protocols on mutation quantification and artifact generation.

Main Results:

• Confirmed the assay's robustness and high confidence limit of detection at approximately 0.6%.
• Identified specific protocols that significantly reduce background error mutations (artifacts).
• Demonstrated a substantial reduction in false positives, facilitating the detection of low-level true mutations.

Conclusions:

• The improved HTS assay offers enhanced accuracy and reliability for mutation detection in biotherapeutic development.
• Optimized protocols are critical for minimizing artifacts and improving the detection of low-frequency mutations.
• This work advances cell line development by providing a more robust and sensitive quality control method.