NEBNext® Ultra™ II Q5® Master

 
The NEBNext Ultra II Q5^® Master Mix is a new formulation of Q5 DNA polymerase that has been optimized for robust, high-fidelity amplification of next-generation sequencing (NGS) libraries. This new formulation further improves the uniformity of amplification of libraries, including superior performance with GC-rich regions.

The polymerase component of the master mix, Q5 High-Fidelity DNA Polymerase, is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 also has the highest fidelity available (> 100-fold higher than that of Taq DNA Polymerase and ~12-fold higher than that of Pyrococcus furiosus (Pfu) DNA Polymerase), resulting in ultra-low error rates.

The NEBNext Ultra II Q5 Master Mix is an aptamer-based hot start formulation that allows convenient room temperature reaction set up. The convenient 2X master mix format contains dNTPs, Mg⁺⁺ and a proprietary buffer, and requires only the addition of primers and DNA template for robust amplification. NEBNext Ultra II Q5 Master Mix is also included in the NEBNext Ultra II DNA Library Prep Kit for Illumina^®. 

NEBNext Ultra II Q5 Master Mix provides improved coverage of known low coverage regions of the human genome.




Libraries were prepared from Human NA19240 genomic DNA. One library was not amplified. The other two libraries were amplified using 5 cycles of PCR with NEBNext Q5 Hot Start HiFi PCR Master Mix (NEB #M0543) or with NEBNext Ultra II Q5 Master Mix (NEB #M0544). Libraries were sequenced on an Illumina® NextSeq® 500. 420 million 75 bp reads were randomly extracted from each dataset, representing an average coverage of 10X. Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4. Reads on each region were counted using bedtools v2.19.1. A: The number of reads overlapping distinct low coverage regions of the human genome (1) are shown for each library. B: From the 420 million 75 bp reads randomly extracted from each dataset, 10X coverage was expected. The % of difficult regions covered at > 10X are shown for each library. The NEBNext Ultra II Q5 Master Mix provides improved coverage of these known low coverage regions, without drop-outs, and shows similar coverage to the unamplified sample.

Ultra II provides the highest and most uniform coverage of difficult sequence regions.




Indexed libraries were prepared from 100 ng of Human NA19240 genomic DNA using a PCR-free workflow or the library prep kits shown, following manufacturers’ recommendations. The PCR-free library was prepared using NEBNext Ultra II. Libraries were sequenced on the Illumina NextSeq® 500. 420 million reads were randomly extracted from each dataset, to produce an average coverage of 10X. Reads were mapped to the GRCh37 reference genome using Bowtie 2.2.4. Reads on each region were counted using bedtools v2.19.1. The number of reads overlapping distinct difficult, low-coverage regions of the human genome (1) are shown for each library. Ultra II provides the highest and most uniform coverage of these difficult regions, and provides the coverage closest to that obtained with a PCR-free protocol.

NEBNext Ultra II Q5 Master Mix provides uniform GC coverage for microbial genomic DNA with a broad range of GC composition.




Libraries were made using 100 ng of the genomic DNAs shown and the NEBNext Ultra II DNA Library Prep Kit. Libraries were amplified using the NEBNext Ultra II Q5 Master Mix, and sequenced on an Illumina MiSeq. 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. NEBNext Ultra II Q5 Master Mix provides uniform GC coverage regardless of the GC content of the DNA.

Stability: Stability testing using up to 30 freeze/ thaw cycles has shown no negative effect on master mix performance. The NEBNext Ultra II Q5 Master Mix may be stored at –20°C for up to 18 months.

Source
An E. coli strain that carries the Q5 High-Fidelity DNA Polymerase gene.

Reaction Conditions
NEBNext Ultra II Q5 Master Mix, DNA template and 1 μM primers in a total reaction volume of 50 μl.

Applications

• Next generation sequencing library construction
• High-fidelity PCR
• Difficult amplification
• High-throughput PCR

Storage Temperature

-20°C 

Companion Products

• NEBNext® Ultra™ II DNA Library Prep Kit for Illumina®
• NEBNext® Ultra™ II End Repair/dA-Tailing Module
• NEBNext® Ultra™ II Ligation Module 
• NEBNext® Ultra™ DNA Library Prep Kit for Illumina®
• NEBNext® DNA Library Prep Master Mix Set for Illumina®
• NEBNext® Singleplex Oligos for Illumina®
• NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1)
• NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 2)
• NEBNext® Multiplex Oligos for Illumina® (Dual Index Primers Set 1)
• NEBNext® DNA Library Prep Reagent Set for Illumina®
• NEBNext® Library Quant Kit for Illumina®

1.    Aird, D. et al. (2011). Analyzing and minimizing PCR amplification bias in Illumina sequencing libraries. Genome Biology. 12(2), R18.