QIAseq Viral Safety and Clearance

The QIAseq xHYB Viral Safety and Clearance solution offers both a non-targeted and targeted approach that combines enzymatic library construction with probe-based hybrid capture to support sensitive detection of viral contaminants in complex biopharmaceutical samples.  

The workflow is compatible with Illumina sequencing platforms and supports multiplexing through Unique Dual Indexes, enabling scalable throughput across process development, cell bank characterization and viral clearance studies. It may be used to generate molecular data to complement in vitro and in vivo assays within a broader, multi-layered viral safety evaluation strategy as described in ICH Q5A(R2). Probe panels can be selected from predefined options, such as Adventitious Agent or MAP/HAP/RAP panels, or customized to align with your viral risk assessment strategy. By enriching targets before sequencing, you gain improved sensitivity in high-background matrices while maintaining flexibility to expand panel content as regulatory expectations and risk profiles evolve. 

Interested in strengthening your viral safety testing strategy? Request a quote or speak with our biopharma specialist team to discuss your application. 

Improved detection in high-background samples

Viral genomes are often present at low copy number relative to host-cell DNA or other background nucleic acids. Because viral genomes are significantly smaller than host genomes, the non-targeted sequencing approach can result in lower sensitivity than the targeted approach, but allows for the detection of all present species. Hybrid capture enrichment increases the proportion of on-target viral reads, improving sensitivity for adventitious agent detection in complex bioprocess matrices. This enrichment-based approach supports sensitive detection of potential viral contaminants in matrices referenced in ICH Q5A(R2), including unprocessed bulk harvests, cell substrates and raw materials of biological origin. 

Mismatch tolerance for diverse and emerging strains

Viral genomes exhibit high mutation rates, which can limit primer-based enrichment approaches. Hybrid capture allows tolerance for sequence mismatches that may occur in non-annotated or emerging strains, supporting broader detection coverage across genetically diverse viral populations. This capability strengthens viral risk assessment when monitoring evolving or unexpected strains. This feature supports risk-based evaluation of viral diversity, consistent with ICH Q5A(R2) recommendations to consider both specific model viruses and potential unknown or emerging agents.

Scalable multiplexing with controlled read allocation

The workflow supports multiplexing via Unique Dual Indexes and is compatible with Illumina platforms. As a starting point, 1 million clusters per sample are recommended for routine analysis, with higher read depth advised for variable viral loads or challenging matrices. This flexibility enables optimization of sequencing depth based on sample type and risk profile.

Consistent library quality

When prepared according to protocol, post-capture libraries exhibit a typical fragment size distribution of approximately 350–450 bp and are compatible with paired-end sequencing on Illumina instruments. Each lot is tested under QIAGEN ISO-certified Quality Management System to ensure consistent product quality and reproducible performance. 

Viral genomes are highly diverse in size, structure and organization. They may consist of DNA or RNA, exist as episomes, integrate into host genomes, or occur in segmented forms, such as those of influenza viruses. This variability complicates comprehensive detection in biopharmaceutical samples, particularly when monitoring for adventitious agents. 

The QIAseq xHYB Viral Safety and Clearance workflow addresses this complexity through offering both a non-targeted and probe-based hybrid capture enrichment approach. ssRNA, dsRNA and dsDNA are converted to double-stranded cDNA and used to generate indexed libraries with the QIAseq FX DNA Library Kit. During this step, mammalian rRNA and Globin RNA are depleted using QIAseq FastSelect Technology.  When performing the non-Targeted QIAseq Universal Metagenome Kit workflow, these libraries are ready for sequencing. 

In the targeted workflow, libraries are then hybridized with biotinylated probes specific to viral targets. Probe–target hybrids are captured using streptavidin-coated beads, washed to remove unbound or loosely bound fragments, and amplified to produce sequencing-ready libraries.

Because hybrid capture tolerates sequence mismatches, it supports the detection of genetically diverse or emerging viral strains that may be missed by strictly primer-dependent methods. Probe content can be scaled to include multiple viral genomes within a single panel, enabling targeted enrichment aligned with defined viral safety strategies. This capability is particularly relevant in the context of ICH Q5A(R2), which emphasizes the need to evaluate potential viral contamination from known and unknown sources, including adventitious agents.

QIAseq xHYB Viral Safety and Clearance is compatible with RNA, DNA or total nucleic acid inputs. Sample types may include cell banks, viral seed stocks, unprocessed bulk harvests and raw materials of biological origin, consistent with matrices referenced in ICH Q5A(R2). When starting with DNA, the workflow can begin directly with library construction using the QIAseq FX DNA Library Kit. When starting with RNA, the workflow includes reverse transcription prior to library preparation.

Converting ssRNA, dsRNA and ssDNA into double-stranded cDNA

For RNA samples, total RNA is first converted into double-stranded cDNA. The reverse transcription product is subsequently converted to double-stranded cDNA and purified using magnetic bead cleanup. The resulting double-stranded DNA serves as the input for library construction. During the first strand synthesis reaction, mammalian host rRNA and Globin RNA are depleted using QIAseq FastSelect technology.

QIAseq FX DNA Library construction

Purified double-stranded DNA is converted into Illumina-compatible sequencing libraries using enzymatic fragmentation, end repair and A-addition in a single-tube reaction. Illumina platform–specific adapters containing Unique Dual Indexes are ligated to both ends of the DNA fragments. Adapter-ligated libraries are purified using magnetic beads to remove excess adapters and are then amplified to generate sufficient material for hybrid capture. When performing the non-targeted workflow, your library is ready to sequence at this step.

Hybrid capture

When performing the targeted workflow, indexed libraries are pooled in equal mass and hybridized overnight with biotinylated QIAseq xHYB probes specific to viral targets. Following hybridization, probe–target complexes are captured using streptavidin-coated magnetic beads. Stringent wash steps remove unbound library fragments, and enriched libraries are then amplified to generate sequencing-ready material.

Analysis

Prepared libraries are compatible with Illumina sequencing platforms using paired-end reads. The resulting FASTQ files can be analyzed using the GeneGlobe Data Analysis portal for viral identification and quantification, or further examined in CLC Genomics Workbench for variant analysis, phylogenetic assessment and evaluation of viral integration sites. Generated sequence data may support documentation of viral detection studies performed as part of a comprehensive viral safety evaluation program.

This is used for applications such as:

• Viral safety risk assessment in biologics manufacturing described in ICH Q5A(R2)
• Adventitious agent testing
• Viral clearance validation studies
• Raw material qualification
• Cell bank and seed stock characterization
• Process development and comparability studies