Product Class: Kit

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

Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering  NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads (NEB #E6177).

Product Introduction

You’ll be thrilled to pieces
 
Do you need a faster, more reliable solution for DNA fragmentation and library construction? The NEBNext® Ultra™ II FS DNA Library Prep Kit meets the dual challenges of constructing high quality libraries from ever-decreasing input amounts, and scalability of library construction.
 
The kit includes a new DNA fragmentation reagent, which is also combined with end repair and dA-tailing reagents, enabling these steps to be performed in the same tube, with no clean-ups or sample loss.
 
You’ll be thrilled to pieces with the result – a reliable, flexible, high-quality library prep that is fast and scalable.
 
  • Perform fragmentation, end repair and dA-tailing with a single enzyme mix 
  • Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content 
  • Prepare high quality libraries from a wide range of input amounts: 100 pg–500 ng 
  • Generate high yields with increased reaction efficiencies and minimized sample loss 
  • Use with DNA in standard buffers (TE, Tris-HCl) and water 
  • Save time with a streamlined workflow: ~ 2.5 hours, with < 15 minutes hands-on time 
  • Vary incubation time to generate a wide range of insert sizes

 
See what others are saying about NEBNext Ultra II FS.

 

 

Catalog # Size Concentration
E7805S 24.0 reactions
E7805L 96.0 reactions

Product Information

Description

The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina provides a fragmentation system: a fast and reliable solution for DNA fragmentation and library construction. A new DNA fragmentation reagent is combined with end repair and dA-tailing reagents, allowing these steps to be performed in the same tube, with no clean-ups or sample loss. The same fragmentation protocol is used for any input amount (100 pg–500 ng), or GC content. The kit also includes the NEBNext Ultra II Ligation Master Mix for adaptor ligation, and the NEBNext Ultra II Q5® Master Mix for uniform, high-fidelity library amplification. Please note that adaptors and primers are not included in the kit and are available separately.

For protocols including bisulfite converted DNA, we recommend the NEBNext Ultra II DNA Library Prep Kit for Illumina.

Features:

  • Fragmentation, end repair and dA-tailing reagents in a single enzyme mix 
  • A single fragmentation protocol, regardless of DNA input amount or GC content 
  • Input amounts: 100 pg–500 ng 
  • Input DNA can be in water, Tris or TE
  • Workflow: ~ 2.5 hours, with < 15 minutes hands-on time 

Download extensive performance data in our technical note. 
 
Also available with optional SPRIselect® beads for size selection/clean-up steps

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.
 
 

Figure 1: NEBNext Ultra II FS DNA produces the highest yields, from a range of input amounts

Libraries were prepared from Human NA19240 genomic DNA using the input amounts and numbers of PCR cycles shown. For NEBNext Ultra II FS, a 20-minute fragmentation time was used. For Kapa™ HyperPlus libraries, input DNA was cleaned up with 3X beads prior to library construction, as recommended, and a 20-minute fragmentation time was used. Illumina® recommends 50 ng input for Nextera, and not an input range; therefore, only 50 ng was used in this experiment. “Covaris®” libraries were prepared by shearing each input amount in 1X TE Buffer to an insert size of ~200 bp using a Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Error bars indicate standard deviation for an average of 3–6 replicates performed by 2 independent users.
Figure 2: NEBNext Ultra II FS DNA provides consistent fragmentation regardless of input amount

Libraries were prepared from Human NA19240 genomic DNA using the input amounts shown. NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. For Kapa™ HyperPlus, input DNA was cleaned up with 3X beads prior to library construction, as recommended, and a 20-minute fragmentation time. Library size was assessed using the Agilent® Bioanalyzer®. Low input (1 ng and below) libraries were loaded on the Bioanalyzer without a dilution. High input libraries were loaded with a 1:5 dilution in 0.1X TE.
Figure 3: NEBNext Ultra II FS DNA provides uniform GC coverage for microbial genomic DNA over a broad range of GC composition

Libraries were prepared using 1 ng of a mix of genomic DNA samples from Haemophilus influenzae, Escherichia coli (K-12 MG1655), Rhodopseudomonas palustris and the library prep kits shown, with 9 PCR cycles for consistency across samples, and sequenced on an Illumina MiSeq®. NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. For Kapa HyperPlus libraries, input DNA was cleaned up with 3X beads prior to library construction, as recommended, followed by a 25-minute fragmentation time. “Covaris” libraries were prepared by shearing 1 ng of DNA  in 1X TE Buffer to an insert size of ~200 bp using a Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). 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.
Figure 4: ULTRA II FS produced 10-15x more DNA library compared with other methods - Data from Peter Ellis of the Wellcome Trust Sanger Institute

Human genomic DNA was subjected to DNA library construction using an existing DNA library construction workflow (CURRENT), NEB Ultra II or NEB Ultra II FS.  Adapter-Ligated libraries were amplified by PCR (4-12 cycles), purified and quantitated using the Agilent Bioanalyzer platform. Values obtained were used to normalize DNA library yield from 12 cycles of PCR.

View additional data  presented at AGBT by Peter Ellis, Senior Staff Scientist at the Wellcome Trust Sanger Institute. 
 

 

This product is related to the following categories:
DNA Fragmentation & RNA Fragmentation Products,
DNA Library Prep for Illumina,
Automation for NEBNext® NGS Library Prep Products,

Kit Components

Kit Components

The following reagents are supplied with this product:

NEB # Component Name Component # Stored at (°C) Amount Concentration
  • E7805S     -20    
      NEBNext® Ligation Enhancer E7374AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AVIAL -20 1 x 0.72 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AVIAL -20 1 x 0.6 ml Not Applicable
      NEBNext® Ultra™ II FS Enzyme Mix E7806AVIAL -20 1 x 0.048 ml Not Applicable
      NEBNext® Ultra™ II FS Reaction Buffer E7807AVIAL -20 1 x 0.168 ml Not Applicable
      TE Buffer (1X) E7808AVIAL -20 1 x 1.1 ml 1 X
  • E7805L     -20    
      NEBNext® Ligation Enhancer E7374AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext® Ultra™ II Ligation Master Mix E7648AAVIAL -20 3 x 0.96 ml Not Applicable
      NEBNext® Ultra II Q5® Master Mix E7649AAVIAL -20 2 x 1.2 ml Not Applicable
      NEBNext® Ultra™ II FS Enzyme Mix E7806AAVIAL -20 1 x 0.192 ml Not Applicable
      NEBNext® Ultra™ II FS Reaction Buffer E7807AAVIAL -20 1 x 0.672 ml Not Applicable
      TE Buffer (1X) E7808AAVIAL -20 1 x 4.3 ml 1 X

Properties & Usage

Materials Required but not Supplied

  • 80% Ethanol (freshly prepared)
  • Nuclease-free water
  • 0.2 ml thin wall PCR tubes
  • NEBNext Singleplex or Multiplex Oligos for Illumina (NEB #E7350, #E7335, #E7500, #E6609, #E7710, #E7730 or #E7600)
  • Magnetic rack or plate (e.g., NEBNext® Magnetic Separation Rack (NEB #S1515S), Alpaqua ® 96S Super Magnet Plate (#A001322), or equivalent)
  • PCR machine
  • Vortex
  • Microcentrifuge
  • For NEB #E7805 only: SPRIselect® Reagent Kit (Beckman Coulter, Inc. #B23317) or AMPure® XP Beads (Beckman Coulter, Inc. #A63881)
  • Optional: 10 mM Tris-HCl, pH 8.0 with 10 mM NaCl (for adaptor dilution of DNA input < 100 ng)

Protocols, Manuals & Usage

Protocols

  1. Protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≤100 ng
  2. Protocol for FS DNA Library Prep Kit (E7805, E6177) with Inputs ≥100 ng
  3. Protocol for FS DNA Library Prep Kit (E7805, E6177) for Large Fragment Sizes (> 550 bp)
  4. 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. When preparing samples using the NEBNext Ultra II FS DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions?
  2. What do I do if I see a precipitate in the Ultra II FS Reaction Buffer?
  3. Following the Fragmentation and End Prep steps of the NEBNext Ultra II FS workflow can the reactions be kept at 4° C?
  4. Do I really need to vortex the Ultra II FS Enzyme Mix?
  5. What type of and how much starting material do I need to use when preparing libraries with the NEBNext Ultra II FS DNA Library Prep Kit
  6. Can I use the NEBNext Ultra II FS DNA Library Prep Kit for bisulfite conversion and EMSeq workflows?
  7. The ligation reaction is very viscous. What will happen if it is not mixed properly?
  8. Can I use the NEBNext Ultra II FS DNA Library Prep Kit to prepare libraries for sequencing on Ion Torrent sequencing instruments?
  9. What is the concentration of my index primers?
  10. Which NEBNext Oligos can be used with this library prep kit?
  11. Can I use this NEBNext kit with adaptors and primers from other vendors than NEB?
  12. Does the fragmentation reagent included in NEBNext Ultra® II FS DNA kits contain NEBNext dsDNA Fragmentase®?
  13. Is the NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® compatible with all sample types?

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 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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