QIAseq xHYB Custom Human Panels

• Flexible custom probe design algorithm allows excellent target region coverage
• Highly sensitive detection of variants with low allele frequencies
• Single-day workflow with hybrid capture flexibility ranging from 30 minutes to overnight
• Automation-friendly workflow

QIAseq xHYB Custom Human Panels provide a flexible customization, hybrid-capture-based solution to enable sample-to-insight, targeted sequencing of DNA by next-generation sequencing (NGS). QIAGEN’s fast and efficient hybridization capture technology facilitates highly uniform enrichment of target sequences and allows comprehensive variant detection with minimal sequencing effort. 

The flexible xHYB workflow allows simultaneous hybridization up to eight indexed whole genome sequencing (WGS) libraries in as little as 30 minutes from as little as 200 ng input per WGS library to yield highly complex libraries.

For customization of enriched targets, QIAGEN offers these options: 

• Fully customized panel: Panel built to target customer’s regions of interest (ROIs)
• Flexibility to use with existing panel: Panel built to be combined with one of the QIAseq xHYB Human Hybrid Capture Panels or another custom panel
• Boost existing panel (no limit on the ROI): Panel built to boost ROIs with additional probes for higher coverage

QIAseq xHYB assays deliver exceptional coverage uniformity regardless of the GC content of the targets in the panel. This uniformity facilitates >99% base-level coverage of targets at ≥20x. Comprehensive and sensitive detection of variants (>98% combined sensitivity for SNVs and indels) is possible while minimizing drop-outs.

Exome and other types of comprehensive genomic profiling (CGP) has been used with increasingly frequency in the past 10 years. CGPs provide an efficient and cost-effective way to screen for disease-associated variants in comparison to WGS. 

Hybrid capture is an efficient target-enrichment method for NGS library contruction. Biotinaylated targeting probes (complementary in sequence to their targets) are designed as baits to capture the targets from a library of DNA molecules. Once the probes are hybridized to their targets, these probe–target hybrids are then bound to streptavidin-coated magnetic beads through interaction with the biotin label on the probes. A magnet is then used to keep these beads with the probe-target hybrids on the side of the tube while the rest of the DNA that is not of interest, is washed away, reducing off-target effects. After washing away unbound DNA, the targets are then amplified and prepared for sequencing.

Generation of indexed WGS libraries

WGS libraries are prepared in the first step prior to enrichment by hybrid capture. We recommend the following WGS library preps (purchased separately), which include QIAseq Unique Dual-Index (UDI) or Combinatorial Dual-Index (CDI) Adapters and are fully compatible with the QIAseq xHYB Custom Human Panel workflow, and allow up to multiplexing 384 samples per sequencing run. Both kits are compatible with genomic DNA derived from a variety of different sample types, such as whole blood, cells, tissues, saliva, FFPE samples and cfDNA. 

QIAseq FX DNA Library Kits: 2.5-hour WGS library prep workflow with enzymatic DNA fragmentation, end-repair and adapter ligation, followed by library amplification.

QIAseq Ultralow Input Kit: for double-stranded DNA that has been fragmented enzymatically, chemically, mechanically or naturally: this kit constructs WGS libraries by end polishing, adapter ligation and library amplification. 

Table 1. Recommended library preparation kits to be used for hybridization capture

Enrichment of ROIs from human genomic DNA

Indexed WGS libraries fragments are bound to biotinylated double-stranded DNA capture probes with highly flexible hybridization times ranging from 30 minutes to overnight incubation. Bound fragments are immobilized on streptavidin beads and non-targeted fragments are washed away. Enriched library fragments are amplified using a proprietary post-capture amplification mix that allows even amplification of DNA regions with vastly different GC contents, which minimizes sequencing bias caused by PCR.

Analysis

Upon completion of sequencing, FASTQ files are uploaded into the QIAGEN CLC Genomics Workbench for filtering, read mapping and variant calling. VCF output is then uploaded into QIAGEN Clinical Insight, which enables a variant filtering cascade that facilitates prioritization of variants for evidence-based interpretation.

QIAseq xHYB Custom Panel may be used in a variety of applications that use targeted sequencing, such as the following:

• Disease-gene identification for rare and inherited disorders
• Population genetics and carrier screening
• Human disease pathways and mechanisms
• Pathogenic infection mechanisms