NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina
Note that Sample Purification beads are not included in this kit. If beads are required, we recommend ordering NEBNext® Ultra™ II FS DNA Library Prep with Sample Purification Beads (NEB #E6177).
You’ll be thrilled to pieces
Do you need a faster, more reliable solution for DNA fragmentation and library construction? The NEBNext® Ultra™ II FS DNA Library Prep Kit meets the dual challenges of constructing high quality libraries from ever-decreasing input amounts, and scalability of library construction.
The 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.
You’ll be thrilled to pieces with the result – a reliable, flexible, high-quality library prep that is fast and scalable.
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
The NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina provides a fragmentation system: a fast and reliable solution for DNA fragmentation and library construction. A new DNA fragmentation reagent is combined with end repair and dA-tailing reagents, allowing 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. The kit also includes the NEBNext Ultra II Ligation Master Mix for adaptor ligation, and the NEBNext Ultra II Q5® Master Mix for uniform, high-fidelity library amplification. Please note that adaptors and primers are not included in the kit and are available separately.
For use with NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) (NEB #E7395), refer to the Protocols tab for UMI Adaptors-specific guidance.
Figure 1: NEBNext Ultra II FS DNA produces the highest yields, from a range of input amounts Libraries were prepared from Human NA19240 genomic DNA using the input amounts and numbers of PCR cycles shown. For NEBNext Ultra II FS, a 20-minute fragmentation time was used. For Kapa™ HyperPlus libraries, input DNA was cleaned up with 3X beads prior to library construction, as recommended, and a 20-minute fragmentation time was used. Illumina® recommends 50 ng input for Nextera, and not an input range; therefore, only 50 ng was used in this experiment. “Covaris®” libraries were prepared by shearing each input amount in 1X TE Buffer to an insert size of ~200 bp using a Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Error bars indicate standard deviation for an average of 3–6 replicates performed by 2 independent users.Figure 2: NEBNext Ultra II FS DNA provides consistent fragmentation regardless of input amount Libraries were prepared from Human NA19240 genomic DNA using the input amounts shown. NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. For Kapa™ HyperPlus, input DNA was cleaned up with 3X beads prior to library construction, as recommended, and a 20-minute fragmentation time. Library size was assessed using the Agilent® Bioanalyzer®. Low input (1 ng and below) libraries were loaded on the Bioanalyzer without a dilution. High input libraries were loaded with a 1:5 dilution in 0.1X TE.Figure 3: NEBNext Ultra II FS DNA provides uniform GC coverage for microbial genomic DNA over a broad range of GC composition Libraries were prepared using 1 ng of a mix of genomic DNA samples from Haemophilus influenzae, Escherichia coli (K-12 MG1655), Rhodopseudomonas palustris and the library prep kits shown, with 9 PCR cycles for consistency across samples, and sequenced on an Illumina MiSeq®. NEBNext Ultra II FS libraries were prepared using a 20-minute fragmentation time. For Kapa HyperPlus libraries, input DNA was cleaned up with 3X beads prior to library construction, as recommended, followed by a 25-minute fragmentation time. “Covaris” libraries were prepared by shearing 1 ng of DNA in 1X TE Buffer to an insert size of ~200 bp using a Covaris instrument, followed by library construction using the NEBNext Ultra II DNA Library Prep Kit (NEB #E7645). Reads were mapped using Bowtie 2.2.4 and GC coverage information was calculated using Picard’s CollectGCBiasMetrics (v1.117). Expected normalized coverage of 1.0 is indicated by the horizontal grey line, the number of 100 bp regions at each GC% is indicated by the vertical grey bars, and the colored lines represent the normalized coverage for each library.Figure 4: ULTRA II FS produced 10-15x more DNA library compared with other methods - Data from Peter Ellis of the Wellcome Trust Sanger Institute Human genomic DNA was subjected to DNA library construction using an existing DNA library construction workflow (CURRENT), NEB Ultra II or NEB Ultra II FS. Adapter-Ligated libraries were amplified by PCR (4-12 cycles), purified and quantitated using the Agilent Bioanalyzer platform. Values obtained were used to normalize DNA library yield from 12 cycles of PCR.
View additional data presented at AGBT by Peter Ellis, Senior Staff Scientist at the Wellcome Trust Sanger Institute.
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Additional Citations
Ellis P, et al. (2021) Reliable detection of somatic mutation in solid tissues by laser-capture microdissection and low-input DNA sequencing. Nat Protoc; 16, 841-871. DOI: 10.1038/s41596-020-00437-6
Real E, et al. (2021) A single-cell atlas of Plasmodium falciparum transmission through the mosquito. Nat Commun; PubMedID: 34045457, DOI: 10.1038/s41467-021-23434-z
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Licenses
This product is licensed for research and commercial use from Bio-Rad Laboratories, Inc., under U.S. Pat. Nos. 6,627,424, 7,541,170, 7,670,808, 7,666,645, and corresponding patents in other countries. No rights are granted for use of the product for Digital PCR or real-time PCR applications, with the exception of quantification in Next Generation Sequencing workflows.
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