Beyond Extreme Sample Conditions: Celemics Solutions for Maximizing NGS Yield from Low-Quality and Low-Amount Samples
The Constraints of Real-World Clinical Samples
One of the most important factors determining the success of a Next-Generation Sequencing (NGS) experiment is the availability of high-quality DNA or RNA.
In research and clinical settings, however, investigators frequently encounter challenging specimens, including highly degraded FFPE tissues stored for extended periods and ultra-low-amount ctDNA isolated from blood.
These suboptimal sample conditions can lead to low library yields and uneven sequencing coverage. In this article, we review internal validation data demonstrating how Celemics Target Enrichment and Library Preparation technologies support reliable data generation under these challenging sample conditions.
1. Stable Library Preparation from Low-Quality FFPE Samples
FFPE samples are essential resources in oncology research, but formalin fixation can cause DNA cross-linking and extensive degradation, reducing Target Capture efficiency. The success of FFPE analysis is therefore influenced by the Library Preparation stage before downstream Target Capture begins.
- Celemics Solution
The Celemics LP Kit is highly optimized to deliver exceptional conversion efficiency and molecular stability, enabling unbiased, uniform library preparation even from heavily damaged FFPE samples with low DIN values. Libraries generated through this optimized chemistry act in perfect synergy with Celemics’ proprietary probe design and hybridization buffer systems, ensuring tight, highly specific capture of even short-fragmented DNA strands.
[Performance Data]
As a result of library preparation using 11 clinical FFPE samples with very low DIN values, Company I’s workflow showed prominent adapter dimer peaks and extensive bubble-like artifact peaks in the high molecular weight regions, resulting in library preparation failure in multiple channels. In contrast, the Celemics Library Prep Kit successfully generated high-quality libraries across all samples, highlighting its superior efficiency and robust performance even with highly degraded FFPE materials.
| Company I Library Prep Kit | Celemics Library Prep Kit |
|---|---|
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* All FFPE clinical samples showed low DIN scores ranging from 1.4 to 2.2, indicating highly degraded DNA input.
2. Validating Mass Multiplexing Stability in FFPE DNA Samples
When scaling up sequencing throughput for low-quality samples, researchers frequently perform multiplexing (pooling) to combine multiple samples into a single sequencing run to control operational costs. However, poor-quality samples often cause high data variance and uneven read allocation during pooling.
- Celemics Solution & Data
To validate multiplexing performance under compromised sample states, an evaluation was conducted using OncoSpan FFPE (Horizon Discovery) reference materials paired with the Celemics CancerScreen Comprehensive – DNA panel. The study compared a single-plex FFPE sample configuration directly against an 8-plex pooled FFPE sample configuration. - Results
The single-plex and 8-plex setups achieved outstanding on-target read ratios of 84.72% and 84.53%, respectively. Furthermore, the standard deviation for sequencing uniformity remained remarkably steady at 0.56 for the single-plex and 0.57 for the 8-plex run. These metrics demonstrate that the Celemics panel guarantees excellent uniformity and highly reproducible data sequencing, completely independent of pooling complexities or library multiplexing density.
| Sample Info. | On-target Read Ratio | Uniformity (St.Dev) |
|---|---|---|
| Single-plex FFPE Sample | 84.72% | 0.56 |
| 8-plex FFPE Sample | 84.53% | 0.57 |
3. Precision Detection of Fusions from Degraded FFPE RNA Samples
Due to its single-stranded nature, RNA degrades far more rapidly than DNA within formalin-fixed matrices. Consequently, accurately identifying structural rearrangements, such as gene fusions (Structural Variation/Fusion) the primary biomarkers for therapeutic oncogenic profiling presents a quite challenging task in clinical NGS workflows.
- Celemics Solution & Data
Celemics addresses this diagnostic barrier through the CancerScreen Comprehensive – RNA panel. The capture performance was strictly evaluated utilizing the Seraseq® FFPE Fusion RNA Reference Material v4 to benchmark somatic cancer-related RNA variant fusion point detection. - Results
Integrative Genomics Viewer (IGV) plot analysis and STAR-Fusion pipeline validation confirmed successful capture and clean data generation for the highly complex LMNA-NTRK1 rearrangement region. These results demonstrate that even within highly fragmented FFPE RNA environments, Celemics targets and isolates critical fusion breakpoints with absolute fidelity by effectively suppressing background noise.
4. Low-Frequency Variant Detection in Ultra-Low-Amount ctDNA Samples
Analyzing ctDNA in Liquid Biopsy workflows requires the detection of low-frequency tumor-derived variants from a very small amount of circulating DNA.
The available sample amount may be extremely limited, and the Variant Allele Frequency(VAF) may fall below 1%, requiring high analytical sensitivity and accurate control of sequencing and amplification errors.
- Celemics Solution & Data
Celemics applies UMI(Unique Molecular Identifier) technology to support stable sequencing depth across target regions when working with ultra-low-amount samples. UMIs assign a unique molecular barcode to individual DNA molecules, allowing PCR duplicates to be identified and errors generated during amplification and sequencing to be corrected during downstream analysis. This approach supports improved capture uniformity and analytical accuracy for low-frequency variant detection.
- Results
The analytical performance of the Celemics ctDNA Panels was evaluated using Seraseq® ctDNA Reference Material v2(AF 1% and AF 0.5%) and Mutation Mix v2 WT.
The panels included:
– Colorectal Cancer Panel: 15 genes
– Breast Cancer Panel: 27 genes
– Lung Cancer Panel: 28 gene markersAcross all cancer panel types, the assays achieved analytical sensitivity above 94% and specificity above 96% for variants at Allele Frequencies of 0.5% and 1.0%.
Conclusion: An End-to-End NGS Workflow for Challenging Samples
For low-quality FFPE DNA, degraded FFPE RNA, and ultra-low-amount ctDNA, increasing sequencing depth alone may not be sufficient to obtain reliable results. The stability of Library Preparation, uniformity under multiplexing conditions, Fusion capture performance, and UMI-based error correction can each influence overall analytical performance. Celemics End-to-End Target Enrichment Workflow integrates Library Preparation, Probe Design, Hybridization Capture, and UMI-based analysis to support consistent and actionable genomic data generation from challenging sample types.
Explore Celemics Target Enrichment Solutions
Are you evaluating NGS workflows for low-quality FFPE tissue, fragmented RNA, or ultra-low-amount ctDNA? Celemics provides End-to-End NGS Solutions from Library Preparation and Target Enrichment to UMI-based error correction tailored to specific sample conditions and research objectives.
