A Novel System Control for Quality Control of Diagnostic Tests Based on Next-Generation Sequencing.

Area of Science:

• Molecular Biology
• Genomics
• Biotechnology

Background:

• A novel system control (SC) was developed for automated AmpliSeq-based next-generation sequencing (NGS) workflows.
• The SC functions as an external positive/sensitivity control, a spike-in quality control (QC) for DNA extraction, and a non-template control for detecting exogenous DNA contamination.

Purpose of the Study:

• To introduce and validate a novel system control (SC) for improving the quality and reliability of NGS-based diagnostic tests.
• To demonstrate the multifaceted utility of the SC in a standard NGS workflow.

Main Methods:

• Plasmids containing wild-type and mutated tobacco mosaic virus sequences were synthesized to create the SC mixture with a 5% variant frequency.
• The SC was used as a stand-alone sample and spiked into clinical samples (formalin-fixed paraffin-embedded and genomic DNA) during NGS runs.

Main Results:

• The SC accurately detected its designed mutations at the expected 5% frequency in a stand-alone sample, with no significant human genome reads.
• Spiking 100 fg of SC into FFPE samples yielded sequencing coverage comparable to 3 fg of genomic DNA, demonstrating sensitivity.
• The SC showed an inverse relationship with DNA input and could indicate insufficient DNA in clinical samples.
• In the presence of PCR inhibitors, SC and target amplicon coverage were eliminated, validating its role as an extraction QC.

Conclusions:

• The developed system control (SC) offers an elegant and comprehensive quality control solution for NGS-based diagnostic applications.
• The SC enhances the robustness and accuracy of NGS data by providing multiple QC functions within a single assay.