The NEBNext® suite of DNA reagents includes dsDNA Fragmentase® (
NEB #M0348) and NEBNext® High-Fidelity 2X PCR Master Mix prepare ( NEB #M0541) to DNA for next generation sequencing. NEBNext® High-Fidelity 2X PCR Master Mix
The NEBNext® High-Fidelity 2X PCR Master Mix (
NEB #M0541) is specifically optimized for the robust, high-fidelity amplification of next-generation sequencing (NGS) libraries, regardless of GC content. The polymerase component of the master mix, Q5™ High-Fidelity DNA Polymerase ( NEB #M0491), is a novel thermostable DNA polymerase that possesses 3´→5´ exonuclease activity, and is fused to a processivity-enhancing Sso7d domain. Q5 High-Fidelity DNA Polymerase also has an ultra-low error rate (> 50-fold lower than that of Taq DNA Polymerase ( NEB #M0273) and 6-fold lower than that of Pyrococcus furiosus (Pfu) DNA Polymerase ( NEB #M0530)). The buffer component of the master mix has been optimized for robust amplification, even with GC-rich amplicons. This combination makes the NEBNext High-Fidelity 2X PCR Master Mix ideal for next generation sequencing library construction.
Indexed libraries were prepared from human IMR90 DNA and split into individual samples for library amplification. Amplification was performed using 8 cycles of PCR with Phusion® High-Fidelity DNA Polymerase, KAPA HiFi™ HotStart ReadyMix or NEBNext® High-Fidelity 2X PCR Master Mix. Libraries were sequenced on an Illumina HiSeq® 2000. To correct for slight differences in the number of aligned reads from each library, 180 million reads were randomly extracted from each dataset, representing an average coverage of ~6X. The number of reads overlapping distinct low coverage regions of the human genome (Aird et.al. Genome Biology, 2011) are shown for each library. The NEBNext High-Fidelity 2X PCR Master Mix gives the most optimal performance of the three enzymes / master mixes tested.
NEBNext® dsDNA Fragmentase®
® dsDNA Fragmentase ® ( NEB #M0348) generates dsDNA breaks in a time-dependent manner to yield 100–800 bp DNA fragments depending on reaction time (1). NEBNext dsDNA Fragmentase contains two enzymes, one randomly generates nicks on dsDNA and the other recognizes the nicked site and cuts the opposite DNA strand across from the nick, producing dsDNA breaks. The resulting DNA fragments contain short overhangs, 5´-phosphates, and 3´-hydroxyl groups. The random nicking activity of NEBNext dsDNA Fragmentase has been confirmed by preparing libraries for next-generation sequencing. A comparison of the sequencing results between gDNA prepared with NEBNext dsDNA fragmentase and with mechanical shearing demonstrates that the NEBNext dsDNA Fragmentase does not introduce any detectable bias during the sequencing library preparation and no difference in sequence coverage is observed using the two methods (2).
Relative size distribution of E. coli DNA fragments with NEBNext dsDNA Fragmentase vs. Nebulization as seen using the Bioanalyzer 2100. The dsDNA Fragmentase sample was incubated for 30 minutes at 37°C with 0.5 µg of DNA per µl of NEBNext dsDNA Fragmentase in 1X Fragmentase Reaction Buffer with 100 µg/ml BSA. The Nebulizer sample was prepared by nebulization of DNA in 50% glycerol for 6 minutes at 35 psi.
®, Fragmentase ® and Phusion ® are registered trademarks of New England Biolabs, Inc.
HiSeq® is a registered trademark of Illumina, Inc.