Is Damaged FFPE DNA Derailing Your NGS? Unlock High-Quality Results with NEBnext FFPE DNA Repair v2 module!

Complete DNA Damage Repair in FFPE Samples: NEBNext® FFPE DNA Repair v2 Module Guide

Is Damaged FFPE DNA Derailing Your NGS? Unlock High-Quality Results with NEBNext® FFPE DNA Repair v2 Module!

Hello, fellow bioscience researchers! 🔬 Today, I've brought some incredibly useful information for those of you working with challenging FFPE (Formalin-Fixed Paraffin-Embedded) samples. While FFPE samples offer excellent preservation, they often suffer from severe DNA damage, making them difficult to use for precise experiments like Next-Generation Sequencing (NGS).

In particular, DNA damage such as deamination can distort base sequences, leading to erroneous analysis results. But don't worry! The NEBNext® FFPE DNA Repair v2 Module can effectively repair damaged DNA, significantly increasing your experimental success rate. In this article, we'll delve into the causes of DNA damage in FFPE samples, the repair mechanisms, and the NEBNext solution.

Why Does DNA in FFPE Samples Get Damaged?

The FFPE (Formalin-Fixed Paraffin-Embedded) process is excellent for preserving tissue indefinitely. However, the formalin used in this process causes critical damage to DNA. Formalin induces DNA strand cross-linking, modifies base pairs, and leads to various types of damage such as deamination, oxidation, nicks, and AP (abasic) sites.

Specifically, deamination is the conversion of cytosine to uracil, which can cause errors in sequencing by misinterpreting uracil as thymine during PCR or NGS. These damages severely reduce the accuracy of genomic analysis, necessitating a repair step before NGS library preparation.

✅ Key Takeaway: Types of FFPE DNA Damage

  • Deamination: Cytosine → Uracil conversion
  • Oxidation: Damage to DNA bases and sugar-phosphate backbone
  • Nicks: Single or double-strand breaks in the DNA
  • AP sites: Locations where a base has been removed

The Remarkable Repair Mechanism of the NEBNext® FFPE DNA Repair v2 Module

The NEBNext® FFPE DNA Repair v2 Module effectively addresses various DNA damages in FFPE samples through a sophisticated enzyme-based repair system.

This module repairs DNA through the sequential action of the following enzymes:

  1. Uracil DNA Glycosylase (UDG/UNG): Accurately recognizes and removes uracil created by deamination in DNA, forming an abasic site.
  2. AP Endonuclease (APE1): Recognizes the abasic site and cleaves the DNA's sugar-phosphate backbone.
  3. DNA Polymerase: Fills the gap with the correct nucleotide (e.g., cytosine instead of uracil).
  4. DNA Ligase: Finally, seals the phosphodiester bond of the DNA strand, completely restoring the damaged section.

This complex yet precise process ensures that FFPE DNA is restored to a stable state, significantly enhancing the efficiency and accuracy of subsequent NGS library preparation.

✅ Summary: DNA Repair Mechanism

  • UDG/UNG removes uracil → APE1 cleaves the backbone → DNA Polymerase fills with the correct base → DNA Ligase seals the gap

FFPE NGS Library Preparation: Key Considerations!

Regarding your inquiry about single-stranded DNA (ssDNA) library preparation, most NGS libraries are ultimately based on double-stranded DNA (dsDNA). Single-stranded DNA is less stable than double-stranded DNA and more prone to errors during manipulation. Therefore, for FFPE samples, it is far more efficient and accurate to first repair the damaged DNA to create stable double-stranded DNA, and then use this as a template for library preparation.

The NEBNext® UltraShear® FFPE DNA Library Prep Kit is optimized for this approach. The main advantages of this kit are:

  • Includes FFPE DNA Repair v2 Module: All enzymes necessary for DNA repair are included in the kit, allowing for immediate experimentation without additional hassle.
  • Non-enzymatic, mild restriction enzyme-based fragmentation: Since FFPE DNA is already damaged, mechanical shearing (e.g., sonication) can cause further damage. This kit uses a gentle enzymatic method for DNA fragmentation, minimizing additional damage.
  • Shearing and library prep before repair: For low-quality DNA samples, fragmenting the DNA before repair and library preparation is advantageous for obtaining higher quality libraries.

If you only require DNA repair, the NEBNext® FFPE DNA Repair v2 Module can be purchased separately and used with your existing library preparation kit. However, given the characteristics of FFPE samples, if you desire the best possible results, we highly recommend using the NEBNext® UltraShear® FFPE DNA Library Prep Kit.

Keywords: FFPE, DNA damage, DNA repair, NGS, library preparation, NEBNext, FFPE DNA Repair v2 Module, deamination, genomic analysis, Formalin-Fixed Paraffin-Embedded, DNA Polymerase

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