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.
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
A separate version of the NEBNext Ultra II FS DNA PCR-free Library Prep Kit isavailable with 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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