Product Class: Kit

NEBNext® FFPE DNA Repair v2 Module

Kits for FFPE DNA library prep are now available, with or without enzymatic DNA fragmentation (NEBNext UltraShear™ FFPE DNA Library Prep Kit NEB #E6655 and NEBNext FFPE DNA Library Prep Kit NEB #E6650)


Catalog #E7360

Product Introduction

This new product further improves repair of FFPE DNA in next generation sequencing (NGS) workflows.

The methods used for fixation and storage of Formalin-Fixed, Paraffin-Embedded (FFPE) DNA samples cause significant damage, making it challenging to obtain high quality sequence data. The NEBNext FFPE DNA Repair v2 Module is an optimized cocktail of enzymes designed to repair FFPE DNA, and supplied with optimized reagents to enable a streamlined workflow for NGS library preparation.  

  • Higher repair efficiency with FFPE DNA
  • A more streamlined workflow for NGS library preparation

Product Information

Description

The NEBNext FFPE DNA Repair v2 Module contains enzymes and buffers that have been optimized to repair FFPE DNA in next generation sequencing workflows in a streamlined manner.

The NEBNext FFPE DNA Repair v2 Module improves upon the performance of the original NEBNext FFPE DNA Repair Mix:

  • Higher repair efficiency with FFPE DNA
  • A more streamlined workflow for NGS library preparation
  • A more convenient reaction buffer containing all required buffer components for both efficient FFPE DNA repair and downstream end repair and dA-tailing.
  • No cleanup is required between repair and library prep, through the use of Thermolabile Proteinase K. 

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 FFPE DNA REPAIR v2 MODULE
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 FFPE DNA Repair v2 Module enables robust library preparation from a broad range of FFPE DNA sample qualities.






Libraries were prepared with 25 ng of Covaris® acoustic-sheared FFPE DNA samples of different qualities and tissue sources. The NEBNext FFPE DNA Repair v2 Module was used, followed by NEBNext Ultra II DNA library preparation (NEB #E7645) with 9 PCR cycles. Libraries were quantified using the Agilent® HS D1000 TapeStation®. The NEBNext FFPE DNA Repair v2 Module improves the yield of FFPE libraries by varying degrees depending on the quality and damage types present in the input DNA. Error bars indicate the standard deviation of two replicates for each library sample.




Figure 3: The NEBNext FFPE DNA Repair v2 Module enables robust library preparation from 5 to 250 ng of FFPE DNA input using both NEBNext hairpin and Unique Dual Index UMI Adaptors




Libraries were prepared with 5, 50, or 250 ng of three different quality normal liver FFPE DNA samples comparing treatment with and without the NEBNext FFPE DNA Repair v2 Module. The NEBNext Ultra II DNA Library Preparation Kit (NEB #E7645) was used with (a) the NEBNext hairpin adaptor and 11, 8 and 6 PCR amplification cycles for 5, 50 and 250 ng inputs, respectively, and (b) the NEBNext Unique Dual Index UMI DNA Adaptors (NEB #E7395) and 11, 8 and 6 PCR cycles for 5, 50 and 250 ng inputs, respectively. All libraries were quantified using the Agilent HS D1000 Tapestation and the average yield of 2 libraries (5 and 50 ng) and 1 replicate (250 ng) was plotted. Error bars indicate standard deviation for the 5 and 50 ng library replicates. The NEBNext FFPE DNA Repair v2 Module is compatible with 5 to 250 ng inputs of FFPE DNA and different NEBNext adaptors.




Figure 4: The NEBNext FFPE DNA Repair v2 Module improves library quality metrics including mapping rate, properly paired reads, and chimeric reads




Libraries were prepared with 50 ng of three different quality normal liver FFPE DNA samples in triplicate, either untreated or treated with the NEBNext FFPE DNA Repair v2 Module before library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq®500. Paired-end reads were downsampled to 1 million reads and mapped to the GRCh38 human reference (RefSeq 884148) using Bowtie2 (v2.3.2). Mapped reads were analyzed with MarkDuplicates (v1.56.0) and Picard SAM/BAM alignment summary metrics (v1.56.0). Treatment with the NEBNext FFPE DNA Repair v2 Module increases the mapping rate and decreases the level of non-properly paired and chimeric reads.




Figure 5: The NEBNext FFPE DNA Repair v2 Module repairs abundant cytosine deamination artifacts present in FFPE DNA samples




50 ng samples of two different normal liver FFPE DNAs (DIN 2.0 and DIN 1.8) were either untreated or treated in duplicate with the NEBNext FFPE DNA Repair v2 Module, followed by library preparation using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq® 500. Paired-end reads were downsampled to 1 million reads and mapped to the GRCh38 human reference (RefSeq 884148) using Bowtie2 (v2.3.2). Mapped reads were analyzed with MarkDuplicates (v1.56.0) and dCdT (CT) mutations were analyzed using Tasmanian (V0.1.3) according to the equation MAX(([C T] - [G A]) / ([NC]+[NG]), EXP(-10)). (a) The frequency of artifactual CT mutations was plotted as a function of read position (0 to 75 bp) in read 1 and read 2. The abundance and positional bias of these artifacts were reduced in two different FFPE DNA samples when treated with the NEBNext FFPE DNA Repair v2 Module. (b) Artifactual mutations identified in read 2 were quantified as an overall frequency. Two replicates for each condition are shown.




Figure 6: The NEBNext FFPE DNA Repair v2 Module repairs oxidative damage in FFPE and non-FFPE DNA samples




A. Libraries were prepared in duplicate with 25 ng of two different normal FFPE DNA samples of different qualities and tissue sources, comparing untreated to treated with the NEBNext FFPE DNA Repair v2 Module. Libraries were prepared using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645), sequenced on the Illumina NextSeq® 500, downsampled to 2 million reads, mapped to the GRCh38 human reference genome using Bowtie2 (v2.3.2). Mapped reads were analyzed by MarkDuplicates (v1.56.0) and Tasmanian (V0.1.3). The frequency of artifactual dGdT (GT) mutations in read 1 are shown for each library replicate. dGdT mutations were calculated according to the equation MAX(([G T] - [C A]) / ([NG]+[NC]), EXP(-10)). 

B. Libraries were prepared in duplicate using 100 ng of human genomic DNA Covaris-sheared in either water* or Tris-EDTA pH 8.0, the NEBNext FFPE DNA Repair v2 Module, and the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Libraries were sequenced on the Illumina NextSeq 500, downsampled to 2 million reads and analyzed for GT mutation frequency as above. *Note: Shearing DNA in water is not recommended by the manufacturer (Covaris) but was used to generate a substrate containing oxidative damage.




Figure 7: The NEBNext FFPE DNA Repair v2 Module improves library yield compared to treatment with UDG enzyme

 

Libraries were prepared in duplicate using 50 ng FFPE DNA input from 4 different normal FFPE DNAs of different tissue sources and qualities (FFPE #1-3 with DIN 2.0 and FFPE #4 with DIN 6.7). Libraries were either untreated, treated with the NEBNext FFPE DNA Repair v2 Module, or treated with UDG enzyme (NEB #M0280) before preparing libraries with the NEBNext Ultra II DNA Library Preparation Kit for Illumina (NEB #E7645). While UDG enzyme can eliminate dU bases from input DNA, the NEBNext FFPE DNA Repair v2 Module can fully repair these damage sites and improve the final library yield (Figures 2, 3, 7) and metrics (Figure 4).




Figure 8: The NEBNext FFPE DNA Repair v2 Module reduces false positives in somatic variant calling arising from cytosine deamination artifacts




Libraries were prepared from 100 ng of four different FFPE DNA samples of varying qualities and tissue sources in duplicate using the NEBNext FFPE DNA Repair v2 Module or NEBNext FFPE DNA Repair Mix (NEB #M6630), the NEBNext Unique Dual Index UMI Adaptors (NEB #E7395), the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645) and 10 PCR cycles. Libraries were captured using a custom cancer hotspot panel (Twist Bioscience®) and sequenced on the Illumina NextSeq 2000. All fastq files were downsampled to the same number of reads (2 X 18 million reads fresh frozen and 2 X 13 million for FFPE). Paired reads were trimmed with fastp (version 0.20.0) and mapped with BWA mem (0.7.17). Markduplicate was processed in picard (2.20.6) with UMI information. UMI information was processed in fgbio (0.8.1) to obtain consensus sequence reads. The final bam files with UMI-based consensus reads were used for somatic variant calling with strelka2 (2.9.10). Somatic variant calls are plotted according to substitution type for each condition. NEBNext FFPE DNA Repair v2 improves the efficiency of cytosine deamination repair (CT/GA) and effectively repairs oxidative damage (GT/CA).
This product is related to the following categories:
FFPE DNA Products,
Next Generation Sequencing Library Preparation Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7360S     -20    
      NEBNext® FFPE DNA Repair Mix v2 E7361AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Thermolabile Proteinase K E7362AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® FFPE DNA Repair Buffer v2 E7363AVIAL -20 1 x 0.168 ml Not Applicable
  • E7360L     -20    
      NEBNext® FFPE DNA Repair Mix v2 E7361AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Thermolabile Proteinase K E7362AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® FFPE DNA Repair Buffer v2 E7363AAVIAL -20 1 x 0.672 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • NEBNext Ultra II DNA Library Prep Kit (NEB #E7645S, #E7645L, #E7103S or #E7103L) for Illumina, or other 
  • 80% Ethanol 
  • Nuclease-free Water 
  • 0.1X TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) 
  • DNase- RNase-free PCR strip tubes 
  • DNA LoBind® Tubes (Eppendorf #022431021) 
  • SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881) 
  • NEBNext Multiplex Oligos for Illumina (www.neb.com/oligos) 
  • Magnetic rack/stand (NEB #S1515, Alpaqua® cat. #A001322, or equivalent) 
  • Thermal cycler 
  • Agilent Bioanalyzer® or TapeStation® and associated reagents and consumables 
  • Adaptor Dilution Buffer NEB #B1430S or NEBNext Unique Dual Index UMI Adaptor Dilution Buffer supplied with NEB #E7395S/L

Protocols, Manuals & Usage

Protocols

  1. Where can I find guidelines and protocols for using FFPE DNA with NEBNext Products?

Manuals

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls.

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. What is the difference between the NEBNext FFPE DNA Repair v2 Module (NEB #E7360) and the NEBNext FFPE DNA Repair Mix (NEB #M6630)?
  2. Will the FFPE DNA Repair v2 Module ligate my DNA fragments?
  3. Will treating my DNA with the FFPE DNA Repair v2 Module hurt my downstream reaction?
  4. Will the FFPE DNA Repair v2 Module blunt the ends of the DNA?
  5. Does the FFPE DNA Repair v2 Module insert random nucleotides into the sequence that it repairs?
  6. Does the FFPE DNA Repair v2 Module remove covalent modifications from DNA bases, such as biotin or digoxigenin?
  7. Does the FFPE DNA Repair v2 Mix repair DNA-protein crosslinks?
  8. Does the FFPE DNA Repair v2 Mix fix blocked 3′ ends?
  9. Can the FFPE DNA Repair v2 Mix repair damage in both single- and double-stranded DNA? Or, does it require double stranded DNA as a template?
  10. If I had a DNA template with mutation sites (i.e. 8-oxoguanine or deaminated cytosines) that are directly adjacent to each other on opposite strands would treatment with the FFPE DNA Repair v2 Mix cause a double strand nick/break?
  11. What gap lengths can be repaired with the FFPE DNA Repair v2 Mix?
  12. Can the FFPE DNA Repair v2 Module repair bisulfite-treated DNA?
  13. When working with fragmented DNA, will the ends be ligated together by the FFPE DNA Repair v2 Module?
  14. How are abasic sites repaired by the FFPE DNA Repair v2 Module?
  15. Can I use the FFPE DNA Repair v2 Module to repair other types of low quality DNA other than FFPE DNA?

Quality, Safety & Legal

Quality Assurance Statement

Quality Control tests are performed on each new lot of NEB product to meet the specifications designated for it. Specifications and individual lot data from the tests that are performed for this particular product can be found and downloaded on the Product Specification Sheet, Certificate of Analysis, data card or product manual. Further information regarding NEB product quality can be found here.

Specifications

The Specification sheet is a document that includes the storage temperature, shelf life and the specifications designated for the product. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]

Certificate Of Analysis

The Certificate of Analysis (COA) is a signed document that includes the storage temperature, expiration date and quality controls for an individual lot. The following file naming structure is used to name these document files: [Product Number]_[Size]_[Version]_[Lot Number]

Safety DataSheets

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

Legal and Disclaimers

Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email busdev@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

New England Biolabs (NEB) is committed to practicing ethical science – we believe it is our job as researchers to ask the important questions that when answered will help preserve our quality of life and the world that we live in. However, this research should always be done in safe and ethical manner. Learn more.

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