NGS Custom Probes for Targeted Sequencing: DNA vs RNA Probes and How to Choose the Right Panel

Whole exome sequencing (WES) and targeted next-generation sequencing (NGS) rely on hybridization capture probes to selectively enrich regions of interest from genomic DNA libraries. The choice of probe chemistry, panel design, and coverage strategy directly determines data quality, variant detection sensitivity, and per-sample cost.

This guide provides a deep technical comparison of DNA and RNA probe technologies, explains the differential capture depth strategy that is reshaping clinical WES economics, and maps Dynegene's full probe portfolio to specific research and clinical applications.

The Science of Hybridization Capture

How NGS Capture Probes Work

In hybridization capture, biotinylated probes complementary to target genomic regions are hybridized with a fragmented DNA library. Probe-target duplexes are then captured using streptavidin-coated magnetic beads, while off-target sequences are washed away. The enriched library is sequenced, providing deep coverage of target regions at a fraction of the cost of whole genome sequencing (WGS).

Two probe chemistries dominate the market: double-stranded DNA probes and double-stranded RNA probes. Each has distinct thermodynamic and practical advantages.

DNA Probes: High Throughput, AI-Optimized Design

Dynegene's Hybridization Capture DNA Probes use high-performance double-stranded DNA probe synthesis technology. The key advantage of DNA probes is their compatibility with high-throughput synthesis on Dynegene's DYHOW platform, which can synthesize up to 4.35 million oligonucleotide sequences on a single chip. This enables rapid custom panel development and cost-effective manufacturing.

The latest generation — the QuarStar Human All Exon Probes 4.0 — incorporates AI model-based probe design algorithms that optimize both capture specificity and uniformity. Combined with proprietary Boosting strategies, these algorithms streamline probe regions while enhancing capture performance and significantly reducing detection costs.

RNA Probes: Superior Sensitivity Through Binding Strength

Dynegene's double-stranded RNA probes exploit a fundamental principle of nucleic acid chemistry: RNA-RNA hybrids have higher binding strength than RNA-DNA or DNA-DNA hybrids. This thermodynamic advantage translates directly into improved capture sensitivity.

Key differentiators of Dynegene's dsRNA probe technology:

• Better sensitivity — Enhanced RNA-RNA hybrid binding strength captures target sequences more efficiently, even from degraded or low-input samples
• Wider coverage — Simultaneous capture of both the sense strand and antisense strand of the target region provides more comprehensive coverage
• Low-frequency mutation performance — The dual-strand capture approach performs well even in detecting low-frequency variants, critical for oncology applications
• Flexible customization — Built on Dynegene's proprietary ultra-high-throughput DNA synthesizer with independent intellectual property rights

Dynegene's Probe Portfolio: Product-by-Product Comparison

DNA Probe Products (QuarStar Series)

QuarStar Human All Exon Probes 4.0 — Standard Version

Parameter	Specification
Probe type	Double-stranded DNA
Genome coverage	~35.5 Mb
Database design	Refseq / CCDS / GENCODE
Applications	Genetic disease testing, precision cancer diagnosis
Customization	Spike-in probes for adaptation to different testing requirements
Hybridization	Rapid and overnight; single-plex and multiplex
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Highlights from the official product page:

• Excellent performance — Outstanding on-target rate and uniformity, enabling more accurate variant detection
• Flexible combination — Spike-in sub-panels adapt to different testing requirements; the high-throughput synthesis platform enables rapid response to customization needs
• Cost advantage — Requires less sequencing data to achieve the same target depth, reducing per-sample testing costs
• Broad compatibility — Works with rapid and overnight hybridization systems; supports multiplexed sample hybridization

QuarStar Human All Exon Probes 4.0 — Tumor Version

Parameter	Specification
Probe type	Double-stranded DNA
Genome coverage	~46 Mb
Additional targets	1,000+ tumor-related genes, fusions, MSI, HRD, HLA
Design technology	AI algorithm-based probe design and Boosting strategies
Minimum hybridization	Ultra-fast: as quick as 30 minutes
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The Tumor version is built on the Standard version and represents Dynegene's most feature-rich exome panel. Its defining innovation is differential sequencing depth — a probe design strategy that applies different capture intensities to different genomic regions based on clinical importance.

Differential Depth Performance (at 15G total sequencing data):

Region Type	Effective Depth
General gene exons	>150x (~190x)
Tumor-related genes	>450x (~500x)
Hotspot regions	>650x (~900x)

This approach allows oncologists to get deep, mutation-sensitive coverage where it matters most — at hotspot genes critical for treatment selection — while maintaining adequate exonic depth for comprehensive variant calling, all within a 15G data budget.

Fusion Gene Detection:

The Tumor version includes enhanced design for fusion hotspot genes and their high-frequency intronic regions:

Fusion Gene	CDS + Intron Coverage
ALK	~900x
ROS1	~800x
RET	~1,000x
NTRK1	~1,000x

Low-frequency fusion detection has been validated down to 2.08% allele frequency (confirmed by ddPCR).

HRD Detection:

The panel selects 23,157 high-frequency heterozygous SNPs, verified by WGS, with balanced GC content and high specificity, enabling precise detection of genomic scars (LOH, TAI, LST) at sequencing depths consistent with general CDS regions.

Additional DNA Probe Panels

Panel	Target Application
QuarStar Liquid Pan-Cancer Panel 3.0	Liquid biopsy pan-cancer screening
QuarStar Pan-Cancer Lite Panel 3.0	Streamlined pan-cancer testing
QuarStar Pan-Cancer Fusion Panel 1.0	Gene fusion detection
QuarStar Pan Cancer Panel 1.0	Comprehensive pan-cancer panel

RNA Probe Products (QuarXeq Series)

QuarXeq Human All Exon Probes 3.0

Parameter	Specification
Probe type	Double-stranded RNA
Database design	Refseq / CCDS / GENCODE
Coverage	Comprehensive CDS regions including TERT promoter
Applications	Genetic disease detection, precision oncology, immunotherapy
Hybridization	Rapid and overnight; single-plex and multiple-plex
Customization	Different panel modules can be spiked in
Product page	detail-316.html

The QuarXeq 3.0 is Dynegene's RNA-based whole exome solution. It accounts for common variations in transcript forms and covers challenging genomic regions such as the TERT promoter. The upgraded probes demonstrate comprehensive improvements in capture efficiency and uniformity.

DNA Probes vs RNA Probes: When to Use Which

Choose DNA Probes (QuarStar) When:

1. Clinical oncology with WES + tumor profiling — The QuarStar 4.0 Tumor version provides differential depth that gives hotspot genes 4–6× deeper coverage than general exons
2. High-throughput clinical labs — DNA probes paired with ultra-fast 30-minute hybridization maximize daily sample throughput
3. Budget-sensitive testing programs — The Standard version requires less sequencing data to achieve target depth
4. Custom panel development — Dynegene's 4.35M oligos-per-chip synthesis platform enables rapid prototyping of spike-in panels

Choose RNA Probes (QuarXeq) When:

1. Maximum capture sensitivity is required — RNA-RNA hybrid binding thermodynamics provide stronger target capture, critical for degraded FFPE samples
2. Low-frequency variant detection — Dual-strand capture (sense + antisense) enhances performance for low allele frequencies
3. TERT promoter analysis — The QuarXeq 3.0 design explicitly includes this challenging region

Compatible Reagent Kits and Workflow

Hybridization Capture Reagent Kits

Reagent Kit	Probe Compatibility
QuarHyb Super DNA Reagent Kit	DNA probes — high performance
QuarHyb DNA Plus 2 Reagent Kit	DNA probes — versatile
QuarHyb DNA Reagent Kit Plus	DNA probes — standard
QuarHyb One Reagent Kit	Streamlined workflows
QuarHyb Super Reagent Kit Pro	RNA probes — optimized

Library Preparation and Accessories

Product	Function
DNA Library Preparation Kit	Library prep for hybridization capture
Fragmentation Reagent	Enzymatic DNA fragmentation
QuarPro Superfast T4 DNA Ligase	High-efficiency adapter ligation
Dynegene Adapter Family	Sequencing adapters
Dynegene Blocker Family	Hybridization blockers
Streptavidin Magnetic Beads	Capture bead selection
iQuars50 NGS Prep System	Automated sample preparation

Frequently Asked Questions

What is the difference between DNA probes and RNA probes for hybridization capture?

DNA probes use double-stranded DNA to capture targets via DNA-DNA hybridization. RNA probes use double-stranded RNA, forming RNA-RNA hybrids with higher binding strength, resulting in more sensitive capture. Dynegene's dsRNA probes capture both sense and antisense strands simultaneously, providing wider coverage and better low-frequency mutation detection. DNA probes (QuarStar series) excel in high-throughput WES and tumor panels; RNA probes (QuarXeq series) are ideal for maximum sensitivity in genetic disease and precision oncology.

What is differential sequencing depth and how does it reduce costs?

Differential depth applies different capture intensities by region importance. At 15G data, QuarStar 4.0 Tumor achieves: >150x for general exons, >450x for tumor genes, >650x for hotspots — delivering deep mutation-sensitive coverage where it matters most without increasing total sequencing data requirements.

Can Dynegene probes detect gene fusions and HRD?

Yes. The Tumor panel includes fusion hotspot intron coverage at ~800–1,000x depth (ALK, ROS1, RET, NTRK1), detecting fusions as low as 2.08% allele frequency (ddPCR-confirmed). HRD detection uses 23,157 WGS-verified, GC-balanced SNPs for LOH/TAI/LST genomic scar analysis.