FFPE sequencing: how to get better data

Overcome the challenges of limited quantity and poor quality DNA obtained from FFPE samples. Now you can increase the discovery of somatic mutations in your NGS analysis of FFPE samples. Learn how to lower failure rates and increase the sensitivity of your assays.

A challenge for NGS analysis of FFPE DNA is assessing low frequency somatic mutations that are key to cancer progression. The small amount and poor quality of DNA from FFPE samples are major obstacles to the discovery of these somatic mutations. Researchers typically experience a 20-25% failure rate in NGS library preparation from FFPE samples.

improved NGS technologies are now available that overcome these limitations for a variety of applications. With our  Swift Biosciences Accel-NGS 2S Hyb DNA Library Kit and Accel-Amplicon Panels you can prepare high complexity NGS libraries with much lower failure rates.

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. 

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.

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.

As formalin fixation time increased, the duplication rate also increased while the achieved depth of coverage decreased. 

FFPE Amplicon Sequencing

Accel-Amplicon Panels can be used with FFPE DNA samples to screen for clinically-relevant mutations on Illumina® platforms. The primer pairs in these panels have been designed for an amplicon size range of 120-160 bp so they are well-suited for short DNA fragments, such as FFPE and cfDNA . The fast, single-tube workflow 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

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.

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