FFPE DNA poses many challenges for library preparation, including low input amounts and highly variable damage from fixation, storage, and extraction methods. Regions of interest are often enriched using hybrid capture-based approaches, and these workflows require a high input of diverse, uniform DNA library.
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. The kit 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.
View or download extensive performance data in our Data Supplement.
Includes FFPE DNA repair reagents plus optimized library prep reagents and protocol
Increased library yields
Improved sequencing metrics
Greater sensitivity of somatic variant calling
5-250 ng input range
Automation-friendly workflow
For further performance and workflow improvement, the NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655) also incorporates specialized enzymatic DNA fragmentation.
View or download extensive performance data in our Data Supplement.
FFPE DNA is often compromised in quality and quantity and is a challenging sample type for library prep and sequencing. The NEBNext FFPE DNA Library Prep Kit addresses these challenges in multiple ways:
Repairs FFPE-induced damage using the NEBNext FFPE DNA Repair Mix v2
Uses NEBNext Ultra II library prep reagents in a protocol optimized for FFPE DNA
Robustly amplifies libraries using NEBNext MSTC™ FFPE Master Mix
High library yields, sufficient for input into target enrichment workflows
User-friendly workflow with minimal hands-on time and multiple safe stopping points
The NEBNext FFPE DNA Library Prep Kit reduces damage from deamination and oxidation due to the fixation, storage, and extraction process for FFPE DNA samples, thereby reducing false positive variant calls.
For further performance improvement, the NEBNext UltraShear FFPE DNA Library Prep Kit (NEB #E6655) also incorporates specialized enzymatic DNA fragmentation.
FIGURE 1: NEBNext FFPE DNA Library Prep Kit workflow
The NEBNext FFPE DNA Library Prep kit has a streamlined workflow with minimal hands-on time. The protocol has been optimized for the user to safely store the reaction after any step in the workflow overnight at -20°C without affecting library yield or quality.
FIGURE 2: The NEBNext FFPE DNA Library Prep Kit enables robust library preparation from a range of sample inputs and quality
Libraries were prepared from 5, 50 or 250 ng of Covaris®-sheared normal tissue FFPE DNA ranging in quality from DNA Integrity Number (DIN) 1.8 to 6.8 with the indicated PCR cycles. Libraries were made in triplicate for 5 and 50 ng input and 1 replicate for 250 ng. Each bar represents the average of triplicates with error bars indicating standard deviation for the 5 and 50 ng inputs. Robust library yields were obtained across sample qualities and input amounts. Most target enrichment workflows require 200 ng of library for hybrid capture input, and sufficient library yield can be obtained using a minimum of 50 ng FFPE DNA with the NEBNext FFPE DNA Library Prep Kit.
Libraries were prepared from 50 ng of Covaris-sheared normal tissue FFPE DNA of either low (DIN 1.8) or high (DIN 6.8) quality using the NEBNext FFPE DNA Library Prep Kit and other library prep kits as shown. Libraries were sequenced on the Illumina® NextSeq® 500 (2 x 76 bases). Libraries were downsampled to 600,000 paired-end reads, mapped using bowtie2 (version 2.3.2.2) to the GrCh38 reference, and duplicates marked using Picard MarkDuplicate (version 1.56.0). The average frequency of C→T mutations at each C position in Read 2 was calculated for two technical replicates using Tasmanian (version 1.0.7). C→T mutations arising from cytosine deamination damage in low quality FFPE DNA are effectively repaired by the NEBNext FFPE DNA Repair v2 mix included in the NEBNext FFPE DNA Library Prep Kit. Other kits show a high level of C→T artifacts in low quality FFPE DNA (DIN 1.8) due to a lack of DNA damage repair.
FIGURE 4: The NEBNext FFPE DNA Library Prep Kit detects expected variants in formalin-compromised reference standard DNA
Libraries were prepared in triplicate from 50, 100, or 250 ng of Covaris-sheared formalin-compromised reference standard DNA (severe) (Horizon Discovery HD803) using either the NEBNext FFPE DNA Library Prep Kit or NEBNext Ultra™ II DNA Library Prep Kit. The full library yield was used in a target enrichment workflow with a custom cancer panel (Twist Bioscience®) as a 6-plex capture reaction. Libraries were sequenced on the Illumina NovaSeq® 6000 (2 x 100 bases). All fastq files were downsampled to 22 million paired-end reads, mapped using BWA mem (version 0.7.17) to the T2T reference, and duplicates marked using Picard MarkDuplicates (version 1.56.0). Variant allele frequencies (VAF) were calculated from Mpileup using Samtools (version 1.16.1) and plotted against the expected VAF in the Horizon reference DNA. A variant with a frequency of 0.88% was missed at 50 ng input for 1/3 replicates, but all variants were detected in all replicates at 100 and 250 ng input. The NEBNext FFPE DNA Library Prep kit improves the correlation of expected to observed VAF in low input libraries (50 ng) compared to standard library prep (NEBNext Ultra II DNA), indicating the benefit of the yield and coverage obtained with the NEBNext FFPE DNA Library Prep Kit including repair with NEBNext FFPE DNA Repair v2.
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