Product Class: Kit

NEBNext UltraExpress® FS DNA Library Prep Kit

Product Introduction

The NEBNext UltraExpress® FS DNA Library Prep Kit is the latest generation of NEBNext DNA library prep, with a fast, streamlined workflow, including enzymatic fragmentation, end prep, and dA-tailing with a single enzyme mix. In under 2 hours, the protocol enables the creation of high yield, high quality libraries, from a broad input amount range, while generating less plastic waste. Designed for simplicity and efficiency, the kit features a single protocol for all input amounts, making NEBNext UltraExpress FS DNA Library Prep well suited for all throughputs.

  • Fast workflow (< 2 hours), including FS "Fragmentation System" enzymatic fragmentation 
  • Fewer steps, requiring only a single reaction tube
  • Fewer clean-up steps required
  • High-quality libraries from a wide input range (10 - 200 ng intact DNA) 
  • Single protocol for all input amounts
  • Automation friendly
Catalog # Size Concentration
E3340S 24.0 reactions
E3340L 96.0 reactions

Product Information

Description

Download the NEBNext UltraExpress FS DNA Library Prep Kit Data Supplement.

The NEBNext UltraExpress FS DNA Library Prep Kit has been developed in response to user need for a faster, streamlined DNA prep workflow. This kit integrates enzymatic fragmentation and delivers high quality libraries from a variety of sample types. It features a single protocol for all DNA inputs, ranging from 10 – 200 ng of intact DNA. The workflow incorporates master mixed reagents, reduced incubation times, and fewer cleanup steps. Use of this kit also generates less plastic consumable waste because the entire library prep is conducted in a single tube. 

  • Go from intact sample to library in under 2 hours with FS (Fragmentation System) enzymatic fragmentation
  • Single-protocol simplicity cuts down on reaction setup time, while streamlined workflows speed up library prep
  • A single-tube workflow means less plastic and consumable waste 
  • Flexibility is enabled with simple guidelines for customized protocols, if desired
  • Automation-friendly protocols for enhanced scalability

Figure 1: NEBNext UltraExpress FS DNA Library Prep workflow

E3340 Workflow



Figure 2: The NEBNext UltraExpress FS DNA Library Prep Kit provides robust library yields over a wide input range

E3340 Library Yield

Libraries were prepared in triplicate from 10, 50, 100 and 200 ng of a 9:1 Human NA19240 genomic DNA (Coriell Institute for Medical Research) and Escherichia coli gDNA (Lofstrand Labs Limited) mixed sample, using the NEBNext UltraExpress FS DNA single-protocol workflow (e.g., same adaptor amount and 6 PCR cycles for all input amounts). Yields exceeded the minimum requirement (40 ng) for a single Illumina® NovaSeq® 6000 run to achieve whole genome sequencing with at least 30X coverage.



Figure 3: The NEBNext UltraExpress FS DNA Library Prep Kit produces libraries with uniform GC coverage and insert size from a range of input amounts

E3340 Input Amounts

Libraries were prepared from 10, 50, 100 and 200 ng of a 9:1 Human NA19240 genomic DNA (Coriell Institute for Medical Research) and Escherichia coli gDNA (Lofstrand Labs Limited) mixed sample, using the NEBNext UltraExpress FS DNA single-protocol workflow (e.g., same adaptor amount and 6 PCR cycles for all input amounts). Libraries were pooled and sequenced on an Illumina NextSeq® 500/550 (2 x 75 bases). Data showed consistent (A) GC coverage and (B) insert size. 2 million paired-end reads from each library were sampled (seqtk v1.0), adaptor-trimmed (seqprep v0.1) and mapped to a composite reference containing GRCh38 and E. coli MG1655 contigs (bowtie2 v2.5.0). GC coverage and insert size distributions were calculated using Picard's CollectGCBiasMetrics and Picard CollectInsertSizeMetrics (v1.56.0); Picard CollectGCBiasMetrics. (1.56) was run on human autosomes only due to the even copy number assumption of the tool. In (A), the horizontal grey line indicates the expected normalized coverage of 1.0, and the dots in shades of green represent read numbers at each GC%. The grey area plot is a histogram representing the distribution of GC content in 100 bp windows of the reference genome.



Figure 4: The NEBNext UltraExpress FS DNA Library Prep Kit produces representative GC coverage and insert size peaks for microbial genomic DNA over a broad range of GC composition

E3340 Sample Types


Libraries were prepared using the NEBNext UltraExpress FS DNA protocol for 10 ng and 100 ng of genomic DNA from Haemophilus influenzae, Escherichia coli, Rhodopseudomonas palustris and Bordetella pertussis. Data showed (A) representative GC coverage and (B) insert size peaks across samples with genome GC contents of 38%-68% GC. Libraries were pooled and sequenced on an Illumina NextSeq 500/550 (2 x 75 bases). 2 million paired-end reads from each library were sampled (seqtk v1.0), adaptor-trimmed (seqprep v0.1), and aligned to their respective reference genomes (bowtie2 v.2.5.0). GC coverage and insert size distributions were calculated using Picard's CollectGCBiasMetrics and Picard CollectInsertSizeMetrics (v1.56.0). The horizontal grey line indicates the expected normalized coverage of 1.0, and the colored dots represent read numbers at each GC%. The grey area plot is a histogram representing the distribution of GC content in 100 bp windows of the reference genome for the 10 and 100 ng inputs.



Figure 5: The NEBNext UltraExpress FS DNA Library Prep Kit provides robust library complexity

E3340 Robust Library Complexity

Libraries were prepared using the NEBNext UltraExpress FS DNA protocol from (A) 10 ng and 100 ng of the ZymoBIOMICS® Microbial Community DNA Standard (Zymo Research®, Catalog #D6306), and (B) 10 ng and 100 ng of the ZymoBIOMICS Microbial Community DNA Standard II (Log Distribution) (Zymo Research, Catalog #D6311). Libraries were pooled and sequenced on an Illumina® MiSeq® (2 x 75 bases). 750,000 paired-end reads from each library were sampled (seqtk v1.3), adaptor-trimmed (seqprep v0.1) and aligned to a composite reference genome (bowtie2 v2.4.5). 1,000 bp windows of constituent genomes were counted (bedtools 2.30.0) and compared across replicates and input levels. High correlation was observed between replicates and between inputs.



Figure 6: The NEBNext UltraExpress FS DNA Library Prep Kit generates libraries representative of input DNA

e3340 Representative Library Input


Libraries were prepared using using the NEBNext UltraExpress FS DNA protocol from 10 ng and 100 ng of the ZymoBIOMICS Microbial Community DNA Standard (Zymo Research #D6306). Libraries were pooled and sequenced on an Illumina MiSeq® (2 x 75 bases). 750,000 paired-end reads from each library were sampled (seqtk v1.3), adaptor-trimmed (seqprep v0.1), and aligned to a composite reference genome (bowtie2 v2.4.5). 1,000 bp windows of constituent genomes were counted (bedtools 2.30.0) and compared across expected and detected composition for both input levels. The detection of specific microbial gDNA was consistent with the expected composition. Expected composition: Cryptococcus neoformans 2%, Saccharomyces cerevisiae 2%, Bacillus subtilis 12%, Escherichia coli 12%, Enterococcus faecalis 12%, Lactobacillus fermentum 12%, Listeria monocytogenes 12%, Pseudomonas aeruginosa 12%, Staphylococcus aureus 12% and Salmonella enterica 12%.



Figure 7: The NEBNext UltraExpress FS DNA Library Prep Kit generates libraries representative of input DNA even with complex mixtures across a log range

E3340 Complex Mixtures

Libraries were prepared using the NEBNext UltraExpress FS DNA protocol from 10 ng and 100 ng of the ZymoBIOMICS Microbial Community DNA Standard II (Log Distribution) (Zymo Research, Catalog # D6311). Libraries were pooled and sequenced on an Illumina MiSeq (2 x 75 bases). 750,000 paired-end reads from each library were sampled (seqtk v1.3), adaptor-trimmed (seqprep v0.1), and aligned to a composite reference genome (bowtie2 v2.4.5). 1,000 bp windows of constituent genomes were counted (bedtools 2.30.0) and compared across expected and detected composition for both input levels. Consistent correlation between expected and detected composition was noted (R2 = 0.96 for 10 ng and R2 = 0.97 for 100 ng). Expected composition: Listeria monocytogenes 89.1%, Pseudomonas aeruginosa 8.9%, Bacillus subtilis 0.89%, Saccharomyces cerevisiae 0.89%, Escherichia coli 0.089%, Salmonella enterica 0.089%, Lactobacillus fermentum 0.0089%, Enterococcus faecalis 0.00089%, Cryptococcus neoformans 0.00089% and Staphylococcus aureus 0.000089%.



 

This product is related to the following categories:
Library Preparation for Illumina Products,
DNA Library Prep for Illumina,
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
  • E3340S     -20    
      NEBNext UltraExpress FS Enzyme Mix E3343AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext UltraExpress® FS Reaction Buffer E3344AVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3345AVIAL -20 1 x 0.48 ml Not Applicable
      NEBNext MSTC High Yield Master Mix E3346AVIAL -20 1 x 1.08 ml Not Applicable
      TE Buffer (1X) E3347AVIAL -20 1 x 0.72 ml 1 X
      NEBNext® Bead Reconstitution Buffer E3348AVIAL -20 1 x 1.92 ml Not Applicable
  • E3340L     -20    
      NEBNext UltraExpress® FS Enzyme Mix E3343AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext UltraExpress® FS Reaction Buffer E3344AAVIAL -20 1 x 0.384 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3345AAVIAL -20 2 x 0.96 ml Not Applicable
      NEBNext MSTC High Yield Master Mix E3346AAVIAL -20 1 x 4.32 ml Not Applicable
      TE Buffer (1X) E3347AAVIAL -20 1 x 2.88 ml 1 X
      NEBNext® Bead Reconstitution Buffer E3348AAVIAL -20 1 x 7.68 ml 1 X

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext UltraExpress® FS DNA Library Prep Kit NEB #E3340)?

Manuals

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

Tools & Resources

Web Tools

FAQs & Troubleshooting

FAQs

  1. When preparing samples using the NEBNext UltraExpress® FS DNA Library Prep Kit, can my input DNA be in EDTA-containing solutions?
  2. What if I see a precipitate in the NEBNext UltraExpress® FS Reaction Buffer?
  3. Do I really need to vortex the NEBNext UltraExpress® FS Enzyme Mix?
  4. The ligation reaction is very viscous. What will happen if it is not mixed properly?
  5. How much starting material must I use when preparing libraries with the NEBNext UltraExpress® FS DNA Library Prep Kit?
  6. What sample types can I use with the NEBNext UltraExpress® FS DNA Library Prep Kit?
  7. Can I use the NEBNext UltraExpress® FS DNA Library Prep Kit for bisulfite conversion and EM-seq™ workflows?
  8. Is it possible to use sheared DNA as input material with the NEBNext UltraExpress® FS DNA Library Prep Kit?
  9. What is the difference between the NEBNext UltraExpress® FS DNA Library Prep Kit and the NEBNext® Ultra™ II FS DNA Library Prep Kit? How do I choose the right one?
  10. Do the NEBNext UltraExpress® Library Prep kits come with beads?
  11. Which NEBNext® Multiplex Oligos (Adaptors & Primers) can be used with the NEBNext UltraExpress Library Prep Kits?
  12. What should I do if I see adaptor dimer in my NEBNext UltraExpress® libraries?
  13. Can this kit be used with adaptors and primers from suppliers other than NEB?
  14. Can I use the NEBNext UltraExpress® FS DNA Library Prep Kit to prepare libraries for sequencing on Ion Torrent instruments?
  15. Does the fragmentation reagent in NEBNext UltraExpress® FS DNA kits contain NEBNext dsDNA Fragmentase®?
  16. Can the NEBNext UltraExpress® FS DNA Library Prep Kit provide sufficient library yields for target enrichment?
  17. When using the NEBNext® FS DNA Library Prep Kit for MGI® (NEB #E9620) or the NEBNext® UltraExpress™ FS DNA Library Prep Kit (NEB #E3340), the ligation reaction is very viscous. What will happen if it is not mixed properly?

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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