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