NGS utilising Formalin Fixed Paraffin Embedded DNA (FFPE)

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 is a major hurdle in discovering somatic mutations for NGS. Swift Biosciences offers sample preparation solutions to overcome these limitations for a variety of applications. Researchers typically experience a 20-25% common failure rate in library preparation of FFPE samples. With Swift’s 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.

Concentration & 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. 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.  For more information, please see the following instructional manual:

NGS Hybridization Capture for Illumina® platforms

Accel-NGS 2S Hyb DNA Library Kit

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 SureSelectXTand SureSelect XT2, 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 (NA1278) 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.

FFPE

Amplicon Sequencing of FFPE DNA

Accel-Amplicon Panels with 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.

FFPE 2

DETECTION OF SOMATIC MUTATIONS IN FFPE SAMPLES