Product Class: Kit

NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®

Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering the NEBNext® Ultra™ II DNA Library Prep with Sample Purification Beads (NEB #E7103)

Product Introduction

Even more from less, for DNA

Are you challenged with trying to obtain higher library yields using ever-decreasing input amounts? Each component in the NEBNext® Ultra™ II DNA Library Prep Kit from NEB has been reformulated, resulting in a several-fold increase in library yield with as little as 500 picograms of input DNA. These advances deliver unprecedented performance, while enabling lower inputs and fewer PCR cycles.

  • Get more of what you need, with the highest library yields
  • Use to generate high quality libraries from a broad range of input amounts, from 500 pg to 1 µg
  • Prepare libraries from ALL your samples, including GC-rich DNA and FFPE DNA samples
  • Improve yields and quality for target enrichment applications
  • Save time with streamlined workflows, reduced hands-on time and automation compatibility, and enjoy the flexibility of kit or module formats

Note: If your input DNA has not already been sheared, we recommend the NEBNext Ultra II FS DNA Library Prep Kit for Illumina, which fragments, end repairs, and dA-tails DNA, all in a single tube with no cleanups or transfers. This kit is available with and without sample purification beads.

Catalog # Size Concentration
E7645S 24.0 reactions
E7645L 96.0 reactions

Product Information

Description


The Ultra II DNA Library Prep Kit for Illumina meets the challenge of constructing high quality libraries from ever-decreasing input quantities. The reagents for each step in the library preparation workflow have been reformulated to enable high yield preparation of high quality libraries from 500 picograms to 1 microgram of input DNA.  This new generation of NEBNext reagents uses a fast, streamlined, automatable workflow and enables use of fewer PCR cycles while also improving GC coverage. The kit is also compatible with PCR-free workflows and is effective with challenging samples such as FFPE DNA.

Now also available with optional SPRIselect® beads for clean-up and size-selection steps.

Advantages
  • Get more of what you need, with the highest library yields 
  • Use to generate high quality libraries  even when you have only limited amounts of DNA, with inputs as low as 500 pg
  • Prepare libraries from ALL of your samples, including GC-rich targets  and FFPE DNA samples 
  • Improve yields and quality for target enrichment applications
  • Save time with streamlined workflows, reduced hands-on time and automation compatibility, and enjoy the flexibility of kit or module formats

To learn more about how NEBNext Ultra II addresses lower input amounts and challenging sample types, download our technical note.

For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) (NEB #E7395), refer to the Protocols tab for UMI Adaptors-specific guidance.

NEBNext Ultra II produces the highest library yields and conversion rates from a broad range of input amounts

A: Libraries were prepared from Human NA19240 genomic DNA using the input amounts and numbers of PCR cycles shown. Manufacturers’ recommendations were followed, with the exception that size selection was omitted.

B: Libraries were prepared from Human NA19240 genomic DNA using the input amounts and library prep kits shown without an amplification step, following manufacturers' recommendations. qPCR was used to quantitate adaptor-ligated molecules, and quantitation values were then normalized to the conversion rate for Ultra II. The Ultra II Kit produces the highest rate of conversion to adaptor-ligated molecules, for a broad range of input amounts.

View additional data on library yield

NEBNext Ultra II provides uniform GC coverage for microbial genomic DNA over a broad range of GC composition and input amounts

Libraries were made using 500 pg, 1 ng and 100 ng of the genomic DNAs shown and the Ultra II DNA Library Prep Kit and sequenced on an Illumina MiSeq®. Reads were mapped using Bowtie 2.2.4 and GC coverage information was calculated using Picard's CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each GC% is indicated by the vertical grey bars, and the colored lines represent the normalized coverage for each library.

View additional data on library quality

NEBNext Ultra II provides superior yields in PCR-free workflows

Libraries were generated from 100 ng of Human NA19240 genomic DNA using the library prep kits shown, following manufacturers' recommendations, and with no amplification step. Library yields were determined by qPCR using the NEBNext Library Quant Kit for Illumina. The NEBNext Ultra II Kit produces the highest yields.


NEBNext Ultra II libraries provide the highest quality sequence data in PCR-free workflows

LIBRARY KIT TOTAL READS % DUPLICATION % MAPPED % CHIMERAS
Ultra II 3,685,029 0.02 97.00 1.10
Kapa Hyper 3,679,136 0.02 97.00 2.00
TruSeq PCR-Free 3,681,643 0.05 96.65 1.38

PCR-free libraries were generated from 100 ng of Human NA19240 genomic DNA using library prep kits shown, following manufacturers’ recommendations, and with no amplification step. Libraries were sequenced on the Illumina NextSeq 500. Reads were mapped to the GRCh37 reference using Bowtie 2.2.4. This data illustrates that NEBNext Ultra II DNA Library Prep Kit enables high quality sequence data in PCR-free workflows, even with low input amounts.

% Mapped: The percentage of reads mapped to human GRCh37 reference. 
% Duplication: The percentage of mapped sequence that is marked as duplicate. 
% Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.

NEBNext Ultra II enables construction of high quality libraries from FFPE DNA samples

FFPE DNA DNA INPUT (ng) LIBRARY YIELDS IN ng % MAPPED % MAPPED IN PAIRS % DUPLICATION % CHIMERAS
Kidney Tumor 17 132 91.5 96.1 0.48 3.0
Lung Tumor 20 232 90.1 94.9 0.42 4.1
Liver Normal 20 691 92.6 94.7 0.33 8.6
Breast Tumor 30 514 91.9 95.1 0.37 4.5

Libraries were prepared from 17–30 ng of human DNA extracted from the FFPE tissue samples listed, amplified using 10 cycles of PCR and sequenced on the Illumina MiSeq.

Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4.
% Mapped: The percentage of reads mapped to Human GRCh37 reference. 
% Mapped in Pairs: The percentage of reads whose mate pair was also aligned to the reference.
% Duplication: The percentage of mapped sequence that is marked as duplicate. 
% Chimeras: The percentage of reads that map outside of a maximum insert size or that have the two ends mapping to different chromosomes.

This data illustrates that the NEBNext Ultra II DNA Library Prep Kit for Illumina enables high quality sequence data, even with low input amounts of FFPE DNA.

View additional data from FFPE DNA samples

NEBNext Ultra II provides superior yields for target enrichment applications.


For Nimblegen® enrichment, libraries were prepared with 100 ng of Human HG02922 genomic DNA, using NEBNext Ultra II with the NEBNext Adaptor and index primers, or the Kapa Library Preparation Kit Illumina Platforms with the SeqCap® Adapter Kit (Roche). Target enrichment was performed with NimbleGen SeqCap Human Exome v3.

For SureSelectXT enrichment, libraries were prepared with 200 ng of Human HG02922 genomic DNA, using NEBNext Ultra II or Kapa Hyper with the NEBNext Adaptor and index primers, or the SureSelectXT Reagent Kit, Illumina (ILM) platforms with Herculase® II Fusion DNA Polymerase. Target enrichment was performed with SureSelect® Human All Exon V6.

Manufacturers’ recommendations were followed, including use of the recommended number of PCR cycles for pre- and post-capture. NEBNext Ultra II libraries resulted in the highest post-capture yields for both enrichment workflows.

View additional data on target enrichment

Lot Control

The lots provided are managed separately and qualified by additional functional validation. Individual reagents undergo standard enzyme activity and quality control assays, and also meet stringent criteria in the additional quality controls listed on each individual component page
This product is related to the following categories:
DNA Library Prep for Illumina,
Chromatin Analysis,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7645S     -20    
      NEBNext® Ultra II End Prep Enzyme Mix E7646AVIAL -20 1 x 0.072 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AVIAL -20 1 x 0.168 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® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml Not Applicable
  • E7645L     -20    
      NEBNext® Ultra II End Prep Enzyme Mix E7646AAVIAL -20 1 x 0.288 ml Not Applicable
      NEBNext® Ultra II End Prep Reaction Buffer E7647AAVIAL -20 1 x 0.672 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® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml Not Applicable

Properties & Usage

Materials Required but not Supplied

  • 80% Ethanol (freshly prepared) 
  • Nuclease-free Water 
  • 0.1X TE (1 mM Tris-HCl, pH 8.0, 0.1 mM EDTA) 
  • DNA LoBind Tubes (Eppendorf #022431021) 
  • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E7600, #E6609, #E7710, or #E7730) 
  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • PCR Machine

For NEB #E7645 only:

  • SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
Optional:
  • 10 mM Tris-HCl, pH 8.0 or 
  • Tris/NaCl, pH 8.0 (10 mM Tris, 10 mM NaCl)
    (for adaptor dilution of DNA input ≤ 100 ng)

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using NEBNext® DNA Library Prep reagents?

Manuals

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

Application Notes

FAQs & Troubleshooting

FAQs

  1. For what type of sample prep/library construction can I use the NEBNext Ultra II DNA Library Prep kit for Illumina?
  2. What type and how much starting material do I need to use when preparing libraries using the NEBNext Ultra II DNA Library Prep Kit for Illumina?
  3. Is size selection required and what is the recommended method?
  4. Can I use the NEBNext Ultra II DNA Library Prep Kit for Illumina reagents for preparing libraries for sequencing on the Ion Torrent PGM or Proton?
  5. Are these reagents available in a customized format, larger format or 96 well formats?
  6. How do I know the concentration of my index primers? 
  7. Which NEBNext Oligos can be used with this library prep kit?
  8. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?
  9. What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer?

Troubleshooting

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.

Licenses

This product is covered by one or more patents.

This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.

For additional information or to inquire about commercial use, please contact busdev@neb.com.

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