Formalin-fixed, paraffin-embedding (FFPE) is a standard method for long term preservation of millions of archived human tissue samples. A challenge for next-generation sequencing (NGS) utilizing FFPE DNA is assessing low frequency somatic mutations that are key to cancer progression, offering desirable biomarkers for ultimately improving clinical outcomes for a wide spectrum of oncology-related diseases. The limited quantity and poor quality of DNA obtained from FFPE samples are major hurdles to the discovery of somatic mutations using NGS. Swift Biosciences offers sample preparation solutions to overcome these limitations for a variety of applications. Researchers typically experience a 20-25% failure rate in library preparation of FFPE samples. With our Accel-NGS® 2S Hyb DNA Library Kit and Accel-Amplicon™ Panels, end users can prepare high complexity NGS libraries with significantly lower failure rates. For extremely damaged DNA, the Accel-NGS 1S Plus DNA Library Kit offers the best solution to rescue precious samples.
SWIFT PRODUCT LINES COMPATIBLE WITH FFPE:
The Swift advantage:
- Compatible with multiple capture technology suitable for a diverse set of panels.
- Highly efficient adapter ligation provides meaningful data from low input samples.
- Exceptional library complexity results in more unique molecules available for capture.
- No adapter titration or heat steps make the kit readily automatable.
The Swift advantage:
- Single-tube assay provides easy and consistent processing.
- Ready-to-sequence libraries in 2 hours.
- 10 ng input DNA required.
- Average amplicon size of 138-149 bp is compatible with FFPE and cfDNA.
- Continguous coverage provides complete coverage of desired regions.
- Coverage uniformity > 95%.
- On target > 95%.
Concentration and Integrity of FFPE DNA
For low quality samples, we recommend quantification by a qPCR method, using both short and long amplicons to accurately determine the concentration and quality of DNA as described in our technical note on Quantification and Quality Assessment of Human DNA Samples. FFPE samples can exhibit varying degrees of DNA damage with adverse consequences that will be more pronounced for amplification of the longer (Alu247) amplicon than the shorter (Alu115) amplicon. Therefore, Alu115 qPCR results accurately detect the total quantity of usable DNA, and the Alu247/Alu115 ratio illustrates the DNA Integrity Score of the sample. High quality DNA is expected to have a DNA Integrity Score of 1.0, while lower quality DNA will have a score of less than 1.0.
NGS Hybridization Capture for Illumina Platforms
The Accel-NGS 2S Hyb DNA Library Kit can be used with FFPE DNA samples to prepare high complexity NGS libraries for hybridization capture. Using this kit, users working with samples of limiting quality or quantity can make libraries for deep sequencing of somatic mutation detection, while saving the sequencing costs associated with whole genome sequencing. A variety of indexing kits allow for compatibility with multiple hybridization capture technologies: Agilent SureSelectXT and SureSelectXT2, NimbleGen™ SeqCap™ EZ, and IDT® xGen® Lockdown® Probes.
Superior Coverage of the Pan-Cancer Panel
The Accel-NGS 2S Hyb Kit and the IDT xGen Pan-Cancer Panel were evaluated from a variety of samples. High quality Coriell DNA (NA12878) performance was compared to DNA samples from formalin-fixed standards and normal kidney FFPE samples that had undergone 6 to 48 hours of formalin fixation. As formalin fixation time increased, the duplication rate also increased while the achieved depth of coverage decreased.
Formalin-Fixed DNA Samples with the Pan-Cancer Panel
Accel-NGS 2S Hyb libraries were constructed from 5 and 1 ng of Horizon Discovery standards. HD701 is not a fixed sample. HD-C749 and HD-C751 are formalin compromised versions of the same DNA present in HD701. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Fixation Time Course with the Pan-Cancer Panel
Accel-NGS 2S Hyb libraries were constructed from 100, 10 and 1 ng of DNA extracted from normal kidney samples that had been frozen or subjected to 6, 24 or 48 hours of fixation. Libraries were enriched with the IDT xGen Pan-Cancer Panel.
Amplicon Sequencing of FFPE DNA
Accel-Amplicon Panels designed for Illumina platforms can be used with FFPE DNA samples to screen for clinically-relevant mutations. Primer pairs within the panels are designed for an amplicon size range of 120-160 bp. For this reason, Accel-Amplicon Panels are well-suited for samples containing short DNA fragments, such as FFPE and cfDNA. The fast, single-tube workflow of Accel-Amplicon Panels requires just 10 ng of input DNA and provides high coverage uniformity and on target percentage.
High Coverage Uniformity for Fresh Frozen and FFPE Samples
An Accel-Amplicon Panel was tested on approximately 10 ng of fresh frozen and FFPE samples. Shown here is the Amplicon coverage, with percentage of targets covered greater than the fraction of the mean indicated in the legend.
Detection of Somatic Mutations in FFPE Samples
The Accel-Amplicon 56G Oncology Panel was used to create libraries from 15 ng of FFPE gDNA. Coverage uniformity and percentage of reads on target were >95%. The average depth of coverage per base ranged 2500-5000X.
Superior Sequencing Data from FFPE Samples of Different Quality
Libraries were generated from FFPE samples using the Accel-Amplicon 56G Oncology Panel with DNA integrity scores defined by an Alu amplicon qPCR assay. DNA input was increased from 10-25 ng with lower quality FFPE DNA.