Impact of Reducing DNA Input on Next-Generation Sequencing Library Complexity and Variant Detection

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• Polymerase chain reaction (PCR) amplification is essential for next-generation sequencing (NGS) library construction.
• While PCR amplifies DNA, it doesn't increase information content, meaning low DNA input can limit assay sensitivity.
• Assessing library complexity and distinguishing unique from duplicate reads is vital for accurate results.

Purpose of the Study:

• To investigate the impact of low DNA input on amplicon-based NGS assays.
• To evaluate the role of unique molecular identifiers (UMIs) in differentiating unique and duplicate reads.
• To understand how library complexity affects variant detection sensitivity.

Main Methods:

• Utilized unique molecular identifiers (UMIs) to track unique DNA molecules during PCR amplification.
• Performed amplicon-based NGS assays with varying low DNA input levels.
• Analyzed sequencing data to assess library complexity, unique coverage depth, and duplicate read proportions.

Main Results:

• Increasing sequencing depth did not consistently improve sensitivity at high levels due to poor correlation between unique and total read coverage.
• Unique coverage depth showed some improvement with increased input but was inconsistent.
• Fluctuations in library complexity led to significant variability in variant allelic fraction estimates between technical replicates.

Conclusions:

• Depth of coverage with unique reads is a critical metric for maintaining sensitivity and accuracy in clinical NGS.
• Solely increasing sequencing depth is insufficient to overcome limitations imposed by low DNA input and variable library complexity.
• Accurate variant detection in clinical NGS requires careful monitoring of unique read depth and library complexity.