Product Class: Kit

NEBNext® Ultra™ II FS DNA PCR-free Library Prep with Sample Purification Beads

The Small size of this product was discontinued on December 15, 2024. The Large size (NEB #E7435L) will continue to be available.

Product Introduction

Don’t be biased! The NEBNext Ultra II FS DNA PCR-free Library Prep Kit for Illumina offers an amplification-free workflow for DNA-seq based on the streamlined and reliable Ultra II FS workflow. Starting with as little as 50 ng of DNA, get the high-quality, high-yield libraries that you need without PCR bias.

  • Includes NEBNext FS (Fragmentation System) enzymatic fragmentation reagents to perform fragmentation, end repair and dA-tailing with a single enzyme mix 
  • Save time with an optimized PCR-free workflow, reduced hands-on time and automation compatibility
  • Generate high quality libraries without an amplification step using inputs from 50 ng to 500 ng of intact DNA
  • Perform fragmentation, end repair and dA-tailing with a single enzyme mix 
  • Benefit from improved library uniformity
  • Use with the NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) or other Illumina-compatible index adaptors with 3′ single T overhang
  • Enjoy the flexibility and reliability of the provided SPRIselect® size selection and clean-up beads
 

Note: For sheared DNA, we recommend the NEBNext Ultra II DNA PCR-free Library Prep Kit for Illumina (NEB#E7410).

 
Catalog # Size Concentration
E7435L 96.0 reactions

Product Information

Description

The NEBNext Ultra II FS DNA PCR-free Library Prep Kit contains the enzymes and buffers required to convert a broad range of intact DNA input amounts into libraries for next-generation sequencing on the Illumina platform, all without an amplification step. The fast, user-friendly workflow includes reliable enzymatic DNA fragmentation, requires minimal hands-on time, resulting in excellent library yields and GC coverage without requiring PCR. 

For added convenience, this kit contains NEBNext Sample Purification Beads (SPRIselect beads from Beckman Coulter) for size selection and enzyme reaction clean-up steps.

Advantages
  • Enjoy flexible input amounts from 50 ng to 500 ng of intact DNA
  • Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content 
  • Perform fragmentation, end repair and dA-tailing with a single enzyme mix 
  • Get more of what you need with reliably high library yields
  • Prepare PCR-free libraries with a broad range of sample types 
  • Save time with streamlined workflows, reduced hands-on time, and automation compatibility
  • Use with the NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) (NEB #E7395) or other Illumina-compatible index adaptors with a 3’ single T overhang
  • Includes sample purification beads for a complete workflow solution

A separate version of the NEBNext Ultra II FS DNA PCR-free Library Prep Kit is available without sample purification beads.

Figure 1: NEBNext Ultra II FS DNA PCR-free Library Preparation Kit generates libraries with higher yields



A. PCR-free libraries were prepared with human cell line NA19240 genomic DNA (Corielle Institute) using NEBNext Ultra II FS DNA PCR-free and Illumina PCR-free library prep kits and size selected for 350 bp long inserts.

B. Libraries of 150-200 bp inserts were prepared using the enzyme-based fragmentation kits NEBNext Ultra II FS DNA PCR-free and Roche Sequencing Kapa HyperPlus PCR-free library prep kits without size selection. NEBNext Unique Dual Index UMI Adaptors DNA Set 1 and Kapa Dual-Indexed Adaptors were used for the NEBNext and Kapa kits, respectively, following manufacturers' recommendations. 



Figure 2: NEBNext Ultra II FS DNA PCR-free Library Prep provides uniform genome coverage with human DNA




NEBNext Ultra II FS DNA PCR-free libraries have more even genome coverage than Illumina DNA PCR-free. Sequencing libraries were prepared according to the manufacturer's recommendations from 250 ng of NA19240 cell line DNA by 2 different users and sequenced on a single NovaSeq® 6000 S4 v1.5 flow cell (PE 2x150 bp). Reads were adaptor trimmed (fastp 0.20.0), aligned to the Telomere-to-Telomere chm13 references (draft 1, bwa-mem 0.7.17), and duplicate marked (samblaster 0.1.24). 2.8B reads were randomly sampled from each bam (sambamba view -s 0.6.8). Coverage of primary, pass-filter assignments was assigned using mosdepth (v0.3.1, -F 772) and plotted for autosomes or the mitochondrial genome (inset). Expected coverage (num_reads*read length/genome size) is indicated. There is significant variation between users for Illumina PCR-free libraries; however, the FS DNA PCR-free libraries are very consistent between users.
This product is related to the following categories:
DNA Library Prep for Illumina,
Automation for NEBNext® NGS Library Prep 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
  • E7435L     Multi-temperature    
      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
      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
      TE Buffer (1X) E7808AAVIAL -20 1 x 4.3 ml 1 X
      DTT E7139AAVIAL -20 1 x 0.5 ml 100 mM
      NEBNext® Sample Purification Beads E6178SVIAL 25 4 x 3.6 ml Not Applicable

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext Ultra II FS DNA PCR-free Library Prep Kit for Illumina?
  2. 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.

FAQs & Troubleshooting

FAQs

  1. What is the main application of the NEBNext Ultra II FS DNA PCR-free Library Prep Kit?
  2. What additional reagents do I need to complete the Ultra II FS DNA PCR-free workflow?
  3. Why is DTT included in the Ultra II FS DNA PCR-free kit? 
  4. Can degraded samples be used for Ultra II FS DNA PCR-free library prep?
  5. What type of and how much starting material do I need when preparing libraries with the NEBNext Ultra II FS DNA PCR-free Library Prep Kit?
  6. When preparing samples using the NEBNext Ultra II FS DNA PCR-free Library Prep Kit, can my input DNA be in an EDTA-containing solution?
  7. What do I do if I see a precipitate in the Ultra II FS Reaction Buffer?
  8. Do I really need to vortex the Ultra II FS Enzyme Mix?
  9. Following the Fragmentation and End Prep steps of the NEBNext Ultra II FS PCR-free workflow, can the reactions be kept at 4˚C?
  10. The ligation reaction is very viscous. What will happen if it is not mixed properly?
  11. Which NEBNext Oligos can be used with NEBNext Ultra II FS DNA PCR-free Library Prep Kit?
  12. Following the Adaptor Ligation step, can the reactions be kept at 4˚C?
  13. Can I use the NEBNext Ultra II FS DNA PCR-free Library Prep Kit for bisulfite conversion or EM-seq™ workflows?
  14. Can I use the NEBNext Ultra II FS DNA PCR-free Library Prep Kit to prepare libraries for sequencing on Ion Torrent sequencing instruments?
  15. How do I quantify my PCR-free libraries?
  16. Can I assess PCR-free library quality on a BioAnalyzer and/or TapeStation?
  17. Can I use this NEBNext PCR-free kit with adaptors from vendors other than NEB?

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]

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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