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<b>NEBNext FFPE DNA Repair v2 </b> <sub><h6>NEB E7360L </h6></sub>

NEBNext FFPE DNA Repair v2
NEB E7360L

NEBNext FFPE DNA Repair v2 Module – 96 reactions; Storage Temp: – 20°C; Shipping: Cool Packs; UN Code: 0000

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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). 

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