NEBNext® Ultra™ II DNA Library Prep

NEBNext Ultra II kits are designed for construction of high-quality libraries using a broad range of input amounts, from pg to μg of DNA. The workflows are fast, streamlined and automatable, with flexible product formats to enable customization.

The original Ultra II DNA Library Kits for Illumina (NEB #E7645, E7103) enable construction of libraries from pre-sheared DNA, with an input range of 500 pg to 1 μg. 


NEBNext Ultra II FS DNA Library Prep – a novel enzymatic fragmentation system 

We have built upon our NEBNext Ultra II DNA library prep workflow to create a fragmentation system, the NEBNext Ultra II FS DNA Library Prep Kit. The Ultra II FS kit includes a new DNA fragmentation reagent, which is also combined with end repair and dA-tailing reagents, enabling these steps to be performed in the same tube, with no clean-ups or sample loss. The same fragmentation protocol is used for any input amount (100 pg–500 ng), or GC content. 

You'll be thrilled to pieces with the result – a reliable, flexible, high-quality library prep that is fast and scalable. 

Highlights of the NEBNext Ultra II FS Kit 

  • Perform fragmentation, end repair and dA-tailing with a single enzyme mix 
  • Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content 
  • Prepare high quality libraries from a wide range of input amounts: 100 pg–500 ng 
  • Generate high yields with increased reaction efficiencies and minimized sample loss 
  • Use with DNA in standard buffers (TE, Tris-HCl) and water 
  • Save time with a streamlined workflow: ~ 2.5 hours, with < 15 minutes hands-on time 
  • Vary incubation time to generate a wide range of insert sizes 
  • Available with optional SPRIselect® beads for size selection/clean-up 


See what others are saying about Ultra II

“In our core, we process large number of samples for genomics applications. Even with the high-throughput Covaris® model, the sample shearing is still a bottle neck for us. The NEBNext DNA DNA kit is such a wonderful product. First of all, it increased our throughput dramatically for DNA sample shearing. The kit combines the shearing, end-repair, dA-tailing, and linker-ligation in a couple simple steps without the need of purification in between. The whole process has been reduced from two days to a few hours. Second, we love the low input option. Because the kit eliminates the need for purification for each of the intermediate steps, it reduces a lot of sample loss during the purification steps, which enables us to use much less starting materials and saves tons on purification beads. Third, we are amazed by the randomness of the enzymatic shearing. We did not observe any significant difference from mechanical shearing. We have used this kit successfully with many large projects including high-throughput genomic screening assays for single cell derived clones.

- Scientist, Cancer research institute

NEBNext Ultra II produces the highest yield libraries from a broad range of input amounts.

Libraries were prepared from Human NA19240 genomic DNA using the input amounts and numbers of PCR cycles shown. Manufacturers’ recommendations were followed, with the exception that size selection was omitted.

NEBNext Ultra II produces the highest rates of conversion to adaptor-ligated molecules from a broad range of input amounts.

Libraries were prepared from Human NA19240 genomic DNA using the input amounts and library prep kits shown without an amplification step, and following manufacturers’ recommendations. qPCR was used to quantitate adaptor-ligated molecules, and quantitation values were then normalized to the conversion rate for Ultra II. The Ultra II kit produces the highest rate of conversion to adaptor-ligated molecules, for a broad range of input amounts.

Read depth correlation shows consistently high coverage for 500 pg–100 ng input amounts.

Libraries were prepared with 100 ng, 1 ng and 500 pg of human NA19240 genomic DNA. Each library was downsampled (sambamba view -s) to include 423 M reads and mapped to GRCh37 using Bowtie 2.2.4. Coverage of each 10 kb region of GRCh37 (as determined by bedtools coverage) was compared between low (500 pg and 1 μg) and 100 ng input. Most regions are covered by ~1,000 reads, as expected. Low and high coverage regions are well correlated.


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Protocols for NEBNext® Ultra™ II DNA Library Prep
Application Notes for NEBNext® Ultra™ II DNA Library Prep
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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.