Product Class: Kit

NEBNext UltraExpress® DNA Library Prep Kit

Product Introduction

The NEBNext UltraExpress DNA Library Prep Kit is the latest generation of NEBNext DNA library prep, with a fast, streamlined workflow. 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. Further, with simplicity at the forefront, the kit features a single protocol for all input amounts, making NEBNext UltraExpress DNA Library Prep well suited for all throughputs.

  • Fast workflow (< 2 hours) 
  • Fewer steps and consumables 
  • Fewer cleanups 
  • High-quality libraries from a wide input range (10 - 200 ng pre-sheared DNA) 
  • Single protocol for all inputs
  • Automation friendly

View or download extensive performance data below and in our Data Supplement.

Catalog # Size Concentration
E3325S 24.0 reactions
E3325L 96.0 reactions

Product Information

Description

View or download extensive performance data below and in our Data Supplement.

Responding to user need for a faster, streamlined DNA prep workflow that delivers high quality libraries from a variety of sample types, the NEBNext UltraExpress DNA Library Prep Kit has a single protocol for all DNA inputs (10 – 200 ng of pre-sheared 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 pre-sheared sample to library in under 2 hours
  • Single-protocol simplicity cuts down on reaction setup time, while streamlined workflows speed up library prep
  • A single-tube workflow means less plastic/consumable waste
  • Flexibility is enabled with simple guidelines for customized protocols, if desired
  • Automation-friendly protocols for enhanced scalability


Figure 1: NEBNext UltraExpress® DNA Library Prep workflow

Workflow diagram



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

Graph of Library Yield

Libraries were prepared from 10, 50, 100 or 200 ng of Human NA19240 genomic DNA (Coriell Institute for Medical Research) using the same adaptor amount and 8 PCR cycles. Yields exceeded the minimum requirement (40 ng) for a single Ilumina® NovaSeq® 6000 run to achieve whole genome sequencing with at least 30X coverage.



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

Graphed Input Amounts

Libraries were prepared from 10, 50, 100 or 200 ng of Human NA19240 genomic DNA (Coriell Institute for Medical Research) using the same adaptor amount and 8 PCR cycles. Libraries were pooled and sequenced on an Illumina® MiSeq® (2 x 75 bases). Data showed consistent GC coverage (A) and insert size (B). 2 million paired-end reads from each library were sampled (seqtk v1.3), adapter-trimmed (seqprep v0.1) and mapped to the GRCh38 reference (bowtie2 v2.4.5), and GC coverage information was calculated using Picard's CollectGCBiasMetrics (v 1.56.0). 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® DNA Library Prep Kit produces representative GC coverage and insert size for a range of sample types


Graph of Sample Type

Libraries were prepared using a single protocol from cell-free DNA (cfDNA, 12 ng) without shearing and Maize DNA (10 ng and 100 ng) sheared to 200bp (Covaris® ME220). Libraries were pooled and sequenced on an Illumina® MiSeq® (2 x 75 bases). 1.4 million paired-end reads from each library were sampled (seqtk v1.3), adapter-trimmed (seqprep v0.1), and aligned to either the GRCh38 reference (human) or Zea mays reference genome (maize) (bowtie2 v2.4.5).

A. All sample types showed representative GC coverage. High- and low-input Maize DNA generated consistent GC coverage. 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.

B. cfDNA insert size had the characteristic fragmentation pattern with 167 bp peak size and periodicity feature of nucleosome-bound DNA in shorter fragments. 10 ng and 100 ng Maize DNA showed consistent insert size, as expected with the shearing protocol used.



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


Graphs of library complexity
Libraries were prepared using a single 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). 1.4 million paired-end reads from each library were sampled (seqtk v1.3), adapter-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® DNA Library Prep Kit generates libraries representative of input DNA


Graphed Input Amounts

Libraries were prepared using a single 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). 1.4 million reads from each library were sampled (seqtk v1.3), adapter-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® DNA Library Prep Kit generates libraries representative of input DNA even with complex mixtures across a log range

Graph of Complex Mixtures

Libraries were prepared using a single 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). 1.4 million reads from each library were sampled (seqtk v1.3), adapter-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
  • E3325S     -20    
      NEBNext UltraExpress® End Prep Reaction Buffer E3324AVIAL -20 1 x 0.056 ml Not Applicable
      NEBNext UltraExpress® End Prep Enzyme Mix E3326AVIAL -20 1 x 0.024 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3327AVIAL -20 1 x 0.24 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E3328AVIAL -20 1 x 0.96 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E3339AVIAL -20 1 x 0.096 ml Not Applicable
      0.1X TE E3341AVIAL -20 1 x 2.4 ml Not Applicable
  • E3325L     -20    
      NEBNext UltraExpress® End Prep Reaction Buffer E3324AAVIAL -20 1 x 0.221 ml Not Applicable
      NEBNext UltraExpress® End Prep Enzyme Mix E3326AAVIAL -20 1 x 0.096 ml Not Applicable
      NEBNext UltraExpress® Ligation Master Mix E3327AAVIAL -20 1 x 0.96 ml Not Applicable
      NEBNext® MSTC™ High Yield Master Mix E3328AAVIAL -20 1 x 3.84 ml Not Applicable
      NEBNext® Bead Reconstitution Buffer E3339AAVIAL -20 1 x 3.84 ml Not Applicable
      0.1X TE E3341AAVIAL -20 1 x 9.6 ml Not Applicable

Properties & Usage

Protocols, Manuals & Usage

Protocols

  1. Where can I find protocols for using the NEBNext UltraExpress® DNA Library Prep Kit (NEB #E3325)?

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. What sample types can I use with the NEBNext UltraExpress® DNA Library Prep Kit?
  2. Is it possible to use enzymatic fragmentation upstream of NEBNext UltraExpress® DNA Library Prep Kit?
  3. What is the recommended protocol for mechanical shearing upstream of NEBNext UltraExpress® DNA Library Prep?
  4. What is the difference between the NEBNext UltraExpress® DNA Library Prep Kit and the NEBNext® Ultra™ II DNA Library Prep Kit? How do I choose the right one?
  5. Do the NEBNext UltraExpress® Library Prep kits come with beads?
  6. Which NEBNext® Multiplex Oligos (Adaptors & Primers) can be used with the NEBNext UltraExpress Library Prep Kits?
  7. What should I do if I see adaptor dimer in my NEBNext UltraExpress® libraries?
  8. What is the best way to quantify my NEBNext UltraExpress® DNA libraries?
  9. Can this kit be used with adaptors and primers from suppliers other than NEB?
  10. What do I do if I see a precipitate in the Ultra II End Prep Reaction Buffer?

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]

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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