NGS from FFPE samples

Formalin-fixed paraffin-embedded (FFPE) tissues are precious treasures for retrospective analysis and are very critical in rare disease analysis. A barrier to the analysis of FFPE samples is that DNA extracted from FFPE tissues is often significantly degraded. Next-generation sequencing (NGS) is the method of choice for genomic analysis, but the artifactual C>T mutations caused by cytosine deamination lead to multiple sequencing errors.  The GeneRead FFPE purification protocol includes a step during which these artifacts are removed enzymatically, thus eliminating  false positives.  Read more about the principle of DNA preparation from FFPE tissue for NGS. 

DNA preparation from FFPE tissue for NGS applications is associated with several challenges. Yields are often limited due to the precious nature of the sample and the compromised status of the DNA. Additionally, artifacts introduced by fixation and embedding conditions, and due to long-term storage, are most prevalent in sequencing results when starting with limited material. One particular problem is the deamination of cytosine bases to deoxyuracil. This leads to a C-T conversion in sequencing reactions.  While the exact mechanism for this is unknown, one explanation is that deamination of cytosine leads to a uracil in that position, which will pair with adenine. Upon sequencing, this modified base will be read as a C>T transition. If strand information is not preserved during library construction, either strand may be sequenced, and therefore, the artifact may appear as either a C>T or a G>A transition. Removal of these artifacts is essential, particularly when used for sequencing analysis of cancer samples, as they may otherwise appear as false-positive mutations. Artifact suppression becomes critical when sequencing FFPE samples, as the relative frequency of false mutations is increased when starting with limited material.

The GeneRead DNA FFPE Kit provides a streamlined procedure for efficient purification of high yields of DNA from small amounts of FFPE tissue sections. Additionally, the procedure includes the removal of deaminated cytosine to prevent false results in DNA sequencing.