FFPE DNA

Archiving of clinical materials as Formalin-Fixed, Paraffin-Embedded (FFPE) samples is a common practice. However, the methods used for fixation and storage severely damage the nucleic acids from these samples. Additionally, FFPE DNA samples are often available in only very small amounts (e.g., a microns-thick tissue section or a region of interest from a laser-capture microdissection). As a result, it can be challenging to construct high quality libraries in sufficient quantity to achieve good sequence data at the desired depth of coverage. FFPE DNA repair is an essential step towards obtaining high-quality libraries from formalin fixed cells and tissues. 
 
The NEBNext^® FFPE DNA Repair V2 Module improves FFPE DNA repair through an optimized combination of enzymes, designed to be used upstream of NGS library prep for the repair of several of the most common types of damage caused by FFPE treatment. The latest version, “V2”, offers higher repair efficiency and a more streamlined workflow than the original NEBNext FFPE DNA Repair Mix. See Figure 1 below for workflow details.
 
The NEBNext UltraShear^™ FFPE DNA Library Prep Kit is the newest option for high-quality library preparation from FFPE-treated samples. This kit incorporates enzymatic fragmentation of FFPE DNA using NEBNext UltraShear, a new enzyme mix optimized for use with FFPE DNA that increases library yields and quality, while improving scalability and ease of use. The kit also repairs damage to DNA caused by the FFPE process and includes reagents and a protocol optimized for library prep of FFPE DNA. The workflow is streamlined and user-friendly, with minimal hands-on time, and the kit improves yields and quality of libraries from a range of FFPE DNA sample input (5-250 ng) and quality. See Figure 2 below for workflow details. NEBNext UltraShear is available as a standalone enzyme mix for enzymatic shearing that is compatible with FFPE DNA. 

For FFPE samples for which enzymatic fragmentation is not desired, the NEBNext FFPE DNA Library Prep Kit is designed for pre-sheared FFPE DNA and improves yields and quality of libraries from a range of sample inputs (5-250 ng) and quality.

 

Table 1: Types of FFPE DNA damage and their ability to be repaired by the NEBNext FFPE DNA Repair v2 Module

FFPE DAMAGE TYPE	Repaired by the NEBNext FFPE DNA Repair V2 Module, NEBNext UltraShear FFPE DNA Library Prep Kit, and NEBNext FFPE DNA Library Prep Kit?
Deamination of cytosine to uracil	Yes
Nicks and gaps	Yes
Oxidized bases	Yes
Blocked 3´ ends	Yes
DNA fragmentation	No
DNA-protein crosslinks	No

Figure 1: Workflow demonstrating the use of the NEBNext FFPE DNA Repair v2 Module with the NEBNext Ultra^™ II DNA Library Prep Kit for Illumina®

Figure 2: The NEBNext UltraShear FFPE DNA Library Prep Kit workflow overview