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NEBNext Ultrashear® FFPE DNA Library Prep Kit

Catalog # Concentration Size List Price Quantity Your Price
E6655L 96 reactions $4,742.00
$4,267.80
E6655S 24 reactions $1,247.00
$1,122.30
Catalog # Size List Price Your Price
E6655L 96 reactions $4,742.00
$4,267.80
E6655S 24 reactions $1,247.00
$1,122.30
Catalog #
Qty:
 
*On-line ordering is for Canadian customers only. Web pricing is applicable only to orders placed online at www.neb.ca

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 Ultrashear™ FFPE DNA Library Prep 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.

View or download extensive performance data in our Data Supplement.

  • Includes specialized enzymatic fragmentation and 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 FFPE DNA library prep without enzymatic fragmentation, the NEBNext FFPE DNA Library Prep Kit (NEB #E6650) is available.

View or download extensive performance data in our Data Supplement.

FFPE DNA is often compromised in quality and quantity and is challenging for library prep and sequencing, including in target enrichment workflows that require high amounts of library input. The NEBNext UltraShear FFPE DNA Library Prep Kit addresses these challenges and improves ease of workflow and scalability:

  • Repairs FFPE-induced damage using the NEBNext FFPE DNA Repair Mix v2
  • Incorporates specialized enzymatic DNA fragmentation using NEBNext UltraShear
  • 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 UltraShear 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.

The combination of specialized enzymatic FFPE DNA fragmentation and repair with optimized reagents and protocols provides superior performance with this challenging sample type.

For FFPE DNA library prep without enzymatic fragmentation, the NEBNext FFPE DNA Library Prep Kit (NEB #E6650) is available.

FIGURE 1: NEBNext UltraShear FFPE DNA Library Prep Kit workflow overview


Image of workflow
The NEBNext UltraShear FFPE DNA Library Prep Kit has a streamlined workflow with minimal hands-on time across a range of inputs from 5-250 ng. 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 UltraShear FFPE DNA Library Prep Kit enables robust library preparation from a range of sample inputs and quality

Image of library yields
Libraries were prepared from 5, 50, or 250 ng of normal tissue FFPE DNA ranging in quality from DNA Integrity Number (DIN) 1.8 to 6.8, with the indicated PCR cycles. Libraries were prepared in triplicate for 5 ng 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 a 200 ng library for hybrid capture input. Sufficient library yield can be obtained using a minimum of 50 ng FFPE DNA with the NEBNext UltraShear FFPE DNA Library Prep Kit.    



FIGURE 3: The NEBNext UltraShear FFPE DNA Library Prep Kit improves library quality

Image showing library yields    
Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear™ FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, using each vendor’s own recommended adaptors (IDT® xGen® EZ UNI, Kapa EvoPlus® Library Prep Kit, QuantaBio® sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). Libraries were sequenced on the Illumina® NovaSeq®6000 (2 x 100 base reads) and downsampled to 5 million paired-end reads. Reads were mapped using Bowtie2 (version 2.3.2.2) to the GRCh38 reference and duplicates were marked using Picard MarkDuplicates (version 1.56.0). Library quality metrics were assessed using Picard Alignment Summary Metrics (version 1.56.0). The level of foldback reads was calculated using Seq_frag_remap (version 0.2). The NEBNext UltraShear FFPE DNA Library Prep Kit improves library quality by reducing the percentage of unmapped, chimeric, non-properly paired, and foldback reads.


FIGURE 4: The NEBNext UltraShear FFPE DNA Library Prep Kit reduces sequencing artifacts

Image showing artifact reduction

Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, with each vendor’s own recommended adaptors (IDT xGen EZ UNI, Kapa EvoPlus Library Prep Kit, QuantaBio sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). Libraries were sequenced on the Illumina NovaSeq 6000 (2 x 100 base reads) and downsampled to 5 million paired-end reads. Reads were mapped using Bowtie2 (version 2.3.2.2) to the GRCh38 reference and duplicates marked using Picard MarkDuplicates (version 1.56.0). The average frequency of C→T mutations at each C position (A) and G→T mutations at each G position (B) in Read 1 and 2 was calculated for two technical replicates using Tasmanian (version 1.0.7). C→T mutations arising from cytosine deamination and G→T mutations arising from oxidative damage in low quality FFPE DNA are effectively repaired by the NEBNext FFPE DNA Repair v2 Mix in the NEBNext UltraShear FFPE DNA Library Prep Kit. Other kits show a high level of C→T and G→T artifacts in low quality FFPE DNA due to a lack of DNA damage repair.



FIGURE 5: The NEBNext UltraShear FFPE DNA Library Prep Kit enables superior on-target coverage in hybrid capture sequencing

Image of hybrid sequencing comps

Libraries were prepared in duplicate from 100 ng of low quality, normal tissue FFPE DNA (DIN 1.8) and 9 PCR cycles, using the NEBNext UltraShear™ FFPE DNA Library Prep Kit. Results were compared to other enzymatic fragmentation-based library prep kits that have been validated for use with FFPE samples, with each vendor’s own recommended adaptors (IDT xGen EZ UNI, Kapa EvoPlus Library Prep Kit, QuantaBio sparQ DNA Library Prep Kit, and Twist Library Preparation EF 2.0 kit). The full library yield was used in singleplex target enrichment with a custom cancer panel (Twist Bioscience®) and libraries were sequenced on the Illumina NovaSeq 6000 (2 x 100 base reads). 15 million paired-end reads were trimmed with Fastp (version 0.20.0) and mapped with BWA mem (version 0.7.17) to the T2T reference. Duplicates were marked using Picard MarkDuplicates (version 2.20.6) with UMI. Target enrichment quality metrics were assessed using Picard HS Metrics (version 2.18.29). The improved yield, coverage, and fraction of usable reads observed in NEBNext UltraShear FFPE DNA Library Prep Kit whole genome sequencing (WGS) libraries correlates to improved coverage, on-target rate, and coverage uniformity in target enrichment libraries.





Reagents Supplied

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-2 -20 1 x 24 reactions
  NEBNext® Sample Purification Beads E6652S 25 1 x 24 reactions
  NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-3 -20 1 x 96 reactions
  NEBNext® Sample Purification Beads E6652L 25 1 x 96 reactions

Properties & Usage

Materials Required but not Supplied

  • 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
  • NEBNext Multiplex Oligos for Illumina® (www.neb.com/oligos)
  • Magnetic rack/stand (NEB #S1515S, 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 NEBNext Unique Dual Index UMI Adaptor DNA Sets (NEB #E7395/E7874/E7876/E7878)


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