Product Class: Kit

NEBNext Ultrashear® FFPE DNA Library Prep Kit


Catalog #E6655

Product Introduction

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.

Product Information

Description

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.





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
  • E6655S     Multi-temperature    
      NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-2 -20 1 x 24 reactions Not Applicable
      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 UltraShear™ Reaction Buffer E6657AVIAL -20 1 x 0.168 ml Not Applicable
      NEBNext UltraShear™ E6656AVIAL -20 1 x 0.096 ml Not Applicable
      500mM DTT E6658AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® MSTC FFPE Master Mix E6651AVIAL -20 1 x 0.6 ml 2 X
      NEBNext® Sample Purification Beads E6652S 25 1 x 24 reactions Not Applicable
      NEBNext® Sample Purification Beads E6652AVIAL 25 1 x 4.3 ml Not Applicable
  • E6655L     Multi-temperature    
      NEBNext Ultrashear® FFPE DNA Library Prep Kit E6655-3 -20 1 x 96 reactions Not Applicable
      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 UltraShear™ Reaction Buffer E6657AAVIAL -20 1 x 0.672 ml Not Applicable
      NEBNext UltraShear™ E6656AAVIAL -20 1 x 0.384 ml Not Applicable
      500mM DTT E6658AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® MSTC FFPE Master Mix E6651AAVIAL -20 2 x 1.2 ml 2 X
      NEBNext® Sample Purification Beads E6652L 25 1 x 96 reactions Not Applicable
      NEBNext® Sample Purification Beads E6652AAVIAL 25 1 x 17 ml Not Applicable

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)

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

Selection Charts

FAQs & Troubleshooting

FAQs

  1. What sample types can I use with the NEBNext UltraShear FFPE DNA Library Prep Kit?
  2. How much starting material is required for the NEBNext UltraShear FFPE DNA Library Prep Kit?
  3. When preparing samples using the NEBNext UltraShear FFPE DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions?
  4. Do I really need to vortex the NEBNext UltraShear enzyme mix before use?
  5. What fragmentation time should be used with the NEBNext UltraShear FFPE DNA Library Prep Kit for FFPE DNA samples?
  6. Is size selection recommended?
  7. What are the safe stopping points in this protocol?
  8. Which NEBNext Multiplex Oligos can be used with the NEBNext UltraShear FFPE DNA Library Prep Kit?
  9. What input amount (nanograms) is recommended for standard versus target enrichment library prep with FFPE DNA samples?
  10. Can I use this NEBNext kit with adaptors and primers from vendors other than NEB?
  11. Will treating my DNA with the FFPE DNA Repair Mix v2 as part of the NEBNext UltraShear FFPE DNA Library Prep Kit hurt my downstream reaction?
  12. Does the FFPE DNA Repair v2 Mix repair DNA-protein crosslinks?
  13. Does the FFPE DNA Repair v2 Mix fix blocked 3′ ends?
  14. 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?
  15. 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?
  16. What gap lengths can be repaired with the FFPE DNA Repair v2 Mix?
  17. Can I fragment my gDNA sample with NEBNext UltraShear® in buffers other than 1X TE (10mM Tris pH 8.0, 0.1mM EDTA)?

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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