The NEBNext Ultra II FS DNA PCR-free Library Prep Kit contains the enzymes and buffers required to convert a broad range of intact DNA input amounts into libraries for next-generation sequencing on the Illumina platform, all without an amplification step. The fast, user-friendly workflow includes reliable enzymatic DNA fragmentation, requires minimal hands-on time, resulting in excellent library yields and GC coverage without requiring PCR.For added convenience, this kit contains NEBNext Sample Purification Beads (SPRIselect beads from Beckman Coulter) for size selection and enzyme reaction clean-up steps.
Advantages
Enjoy flexible input amounts from 50 ng to 500 ng of intact DNA
Experience reliable fragmentation with a single protocol, regardless of DNA input amount or GC content
Perform fragmentation, end repair and dA-tailing with a single enzyme mix
Get more of what you need with reliably high library yields
Prepare PCR-free libraries with a broad range of sample types
Save time with streamlined workflows, reduced hands-on time, and automation compatibility
Use with the NEBNext Multiplex Oligos for Illumina (Unique Dual Index UMI Adaptors DNA Set 1) (NEB #E7395)or other Illumina-compatible index adaptors with a 3’ single T overhang
Includes sample purification beads for a complete workflow solution
A separate version of the NEBNext Ultra II FS DNA PCR-free Library Prep Kit isavailable without sample purification beads.
Figure 1: NEBNext Ultra II FS DNA PCR-free Library Preparation Kit generates libraries with higher yieldsA. PCR-free libraries were prepared with human cell line NA19240 genomic DNA (Corielle Institute) using NEBNext Ultra II FS DNA PCR-free and Illumina PCR-free library prep kits and size selected for 350 bp long inserts.B. Libraries of 150-200 bp inserts were prepared using the enzyme-based fragmentation kits NEBNext Ultra II FS DNA PCR-free and Roche Sequencing Kapa HyperPlus PCR-free library prep kits without size selection. NEBNext Unique Dual Index UMI Adaptors DNA Set 1 and Kapa Dual-Indexed Adaptors were used for the NEBNext and Kapa kits, respectively, following manufacturers" recommendations.Figure 2: NEBNext Ultra II FS DNA PCR-free Library Prep provides uniform genome coverage with human DNANEBNext Ultra II FS DNA PCR-free libraries have more even genome coverage than Illumina DNA PCR-free. Sequencing libraries were prepared according to the manufacturer"s recommendations from 250 ng of NA19240 cell line DNA by 2 different users and sequenced on a single NovaSeq®6000 S4 v1.5 flow cell (PE 2x150 bp). Reads were adaptor trimmed (fastp 0.20.0), aligned to the Telomere-to-Telomere chm13 references (draft 1, bwa-mem 0.7.17), and duplicate marked (samblaster 0.1.24). 2.8B reads were randomly sampled from each bam (sambamba view -s 0.6.8). Coverage of primary, pass-filter assignments was assigned using mosdepth (v0.3.1, -F 772) and plotted for autosomes or the mitochondrial genome (inset). Expected coverage (num_reads*read length/genome size) is indicated. There is significant variation between users for Illumina PCR-free libraries; however, the FS DNA PCR-free libraries are very consistent between users.
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