FFPE Use in Next-Generation Sequencing

Overview

They are finding NGS next-generation sequencing analytical methods on DNA derived from FFPE clinical material is proving to be a powerful component in both oncological research and clinical diagnostics. Fresh- or snap-frozen samples are an excellent source for DNA, but they’re costly to collect and maintain and therefore have limited availability. This makes large scale retrospective studies of tumor-related cancers problematic, thus providing an open door for FFPE. Such studies typically give information regarding possible therapeutic targets as well as crucial prognostic information.  

Although there are many valid questions regarding the viability of FFPE, several studies have established that NGS can be performed using DNA from FFPE tissue successfully. The accuracy and reproducibility of such analyses, as well as their robustness to the bio-molecular quality of the samples used, is still the subject of many ongoing clinical studies. 

Comparative studies find that reasonable consistency of gene expression quantified from FFPE and FF specimens likely attributed the oligonucleotide probes measure expression being located at several positions across a gene. In addition to mRNA transcript quantification, microarray technology has been adapted to measure DNA copy number, single nucleotide polymorphisms (SNPs), and DNA methylation. 

Developing a Sequence Library from FFPE Samples

The preparation of the sequencing library is the first step in the NGS process. Many research outfits deploy different approaches in preparing a sequencing library. The variables in set-ups depend much upon the sequencing platform and the planned analysis (whole-genome sequencing, whole-exome sequencing, targeted DNA sequencing, whole-transcriptome sequencing, targeted RNA sequencing, ChIP-seq, RIP-seq, epigenetic studies.  

A sequencing library can be made by starting from genomic DNA or RNA. The workflow for the preparation of a DNA sequencing library consists of addressing the following areas:

  • Fragmentation and Sizing 

  • Attachment of the Adapters

  • Quantification 

The Benefits of Using FFPE Samples for Sequencing

Formalin fixed-paraffin-embedded (FFPE) tissue processing remains the most economical approach for long-term tissue specimen storage. Therefore, the potential to use FFPE preserved specimens for high throughput genomic applications to FFPE specimens is an active point of research to determine how accurate the analytical method is in practice. 

Clinical studies reveal certain advantages of working with FFPE versus Frozen in sequencing applications: 

Challenges with RNA FFPE samples

 The use of formalin-fixed paraffin-embedded (FFPE) samples offers unique challenges:

  • Acquiring high-quality sequencing data due to the wide distribution of sample quality

  • Differences in formalin fixation methods

  • Differences in storage conditions

  • Age of FFPE samples which may lead to cross-linked and/or degraded nucleic acid and inconsistent extraction yield

  • Insufficient DNA integrity or the presence of PCR inhibitors can lead to low read depth and allele dropout

  • Usage of FFPE specimens for ChIP-seq difficulties due to limited isolation of soluble DNA-protein complexes altered by excessive chemical cross-linking during the formalin-fixation process 

Best Practices for Using FFPE in Analytical Sequencing 

Because of the noted challenges using FFPE for various sequencing technology, the FDA weighs in harvesting methods and guidelines to help ensure a viable product. The National Cancer Institute (NCI) has published evidence-based best practices for FFPE DNA extraction, and the European Committee for Standardization Technical Specifications have also published a reference to the pre-analytical phase of FFPE sampled DNA.  

Tissue Procurement

The location of the biopsy and the amount of tissue harvested are of specific importance. These factors can influence the prediction of copy number variations. Despite the recent technical advances of NGS, tissue sample quality, and large amounts of DNA required often limit the sequencing process. 

Although it is essential for pathologists to understand the amount of tissue needed, it is likely even more important for the radiologist or surgeon to perform the procedure to acquire the tissue to understand. This makes communicating about the specific tissue requirements between the clinician and pathologist before the tissue harvest surgery an essential part of the success process. 

*The minimum quantity of DNA requested by most sequencing laboratories is between 50 and 200 ng. 

Comparable Success in Various Sequencing Methodologies Using FFPE vs. FF

Since miRNAs are more stable than RNA molecules, HTS is quite promising for quantifying miRNA profiles from FFPE specimens. These studies have found that miRNA-seq data generated from FFPE specimens have a similar number of total reads with just a slightly shorter average read length after trimming for adapter sequences.

DNA-seq has been modified to measure global DNA methylation patterns that are almost identical to methylation arrays using bisulfite treatment of DNA. Although less popular than DNA and RNA-seq, the use of FFPE in bisulfite sequencing has demonstrated a viable result. 

Conclusion

Multiple studies cite supportive data that accurate SNV can be identified from DNA-seq data from FFPE specimens. Despite the apparent concerns of FFPE specimen quality, it is widely accepted that paired with DNA-seq technology, it still holds value as it could provide the necessary data in order to fuel new therapeutic discoveries.

For more information on how Geneticist can help you procure large quantities of FFPE samples for your advanced sequencing project, please contact us.