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