Guide to Quality Control for Viral Sequencing

Viral sequencing has evolved into a sophisticated tool, empowering researchers to unravel the intricacies of viral genomes and their implications on human health. As we delve into the realm of viral genomics, meticulous quality control (QC) steps become indispensable to ensure the fidelity and accuracy of sequencing data. In this comprehensive guide, we will delve into the intricacies of QC measures at distinct stages of viral sequencing, including advanced techniques and technology details.

Read our article about The Workflow of Viral Metagenomics.

Evaluating the Viral RNA Extraction Process

The cornerstone of a resilient viral sequencing protocol lies within the meticulous extraction of superior-grade viral RNA. This procedural stage remains susceptible to inherent variations stemming from disparate sample types, extraction methodologies, and plausible contaminants. A stringent quality control regimen serves to safeguard the unblemished integrity and untarnished purity of the procured RNA specimens:

• RNA Integrity Analysis: Employ cutting-edge microfluidic electrophoresis platforms, exemplified by the likes of Bioanalyzer or TapeStation, to yield RNA Integrity Numbers (RIN). Elevated RIN values, approximating the pinnacle of 10, signify nominal degradation, thereby upholding the veritable representation of viral RNA.
• Quantitative PCR (qPCR): Deploy quantitative polymerase chain reaction (qPCR) to gauge the RNA concentration, thereby assuring the availability of adequate material for ensuing downstream methodologies. Additionally, undertake a quantification of residual DNA contamination, a potential jeopardizing factor capable of impinging upon the veracity of outcomes.

Quality Control of Viral DNA or cDNA

In the realm of library preparation workflows, it is a common necessity to fragment DNA or cDNA. A pivotal step in this process involves conducting a meticulous quality assessment to ensure that the fragmented DNA aligns with the appropriate size parameters before advancing further.

• Fragmentation of DNA or cDNA: This step involves breaking down the DNA or cDNA molecules into smaller fragments. It is a crucial step in library preparation for sequencing.
• Verifying Size: Subsequent to the fragmentation process, a critical stratagem involves the meticulous validation of whether the resultant fragments harmonize with the anticipated size spectrum. The efficaciousness of the impending sequencing endeavor finds its underpinning in the veracity of this particular procedural facet. The visual elucidation of the size distribution of the fractured nucleic acid entities finds realization through the agency of microfluidic electrophoresis platforms, typified by the likes of the Bioanalyzer.
• Visualizing Size Profile: The Bioanalyzer, in conjunction with analogous apparatuses, manifests as a tool par excellence in depicting the dimensional distribution of the DNA or cDNA fragments. These instrumentalities bestow the capability to apprehend the size spectrum and contour of the fragments, thus corroborating their adherence to the stipulated size parameters.
• Purification if Necessary: If the fragments are not in the correct size range, the sample may need to be purified to remove unwanted or improperly sized fragments.
• Cross-Contamination Concerns: The use of classical electrophoresis for visualization and extraction of the region of interest can lead to a risk of cross-contaminations between samples. Hence, modern systems utilize separate channels for each sample, eliminating the possibility of cross-contamination during analysis.
• Confirming Fragment Size: Once the correct size range is confirmed, the next step is to verify the fragment size of the DNA or cDNA samples. Some systems allow for accurate analysis of fragment sizes and provide high-resolution visualization.
• Sample Conservation for Sequencing: The use of the analysis systems requires only a small volume of the original sample (1-2 uL). This conservation of the original sample volume ensures that there is enough material left for the sequencing process.

Quality Control in Viral Mutation Detection

Unveiling viral mutations mandates meticulous QC strategies, particularly in the era of emerging variants:

• Ultra-Deep Sequencing: Harness deep sequencing with high coverage across the viral genome. Analyze the frequency and distribution of mutations using tools like VarScan or GATK, thus enhancing sensitivity in mutation detection.
• Reference Material Validation: Integrate well-characterized reference materials harboring known mutations. Validate your pipeline's ability to detect mutations accurately using these controls, ensuring a reliable foundation for mutation analysis.

Quality Control in Virus Detection

The ultimate goal of viral sequencing is precise virus detection. Unveil this realm with a fusion of conventional and cutting-edge QC methodologies:

• Digital Microfluidics: Leverage digital microfluidics to compartmentalize and amplify single viral molecules. This approach amplifies sensitivity, enabling the detection of low-titre viral samples.
• Sensitivity and Specificity Analysis: Establish sensitivity and specificity benchmarks using clinical isolates with varying viral loads. ROC curve analysis quantifies the assay's ability to discriminate true positives from false positives.

Summary

Virus sequencing plays a crucial role in not only detecting but also in tracking, monitoring, and identifying new variants. The utility of next-generation sequencing (NGS) spans across multiple established applications. Ensuring the reliability of sequencing outcomes hinges on meticulous quality control steps. These steps are vital in assessing the soundness, molecular weight, and concentration of initial nucleic acids and libraries. Whether handling modest or extensive workloads, these systems contribute significantly to attaining consistent and effective sample quality control (QC). This compilation draws from diverse sources and literature searches.