DNA Fragmentation
See below for our NEBNext Enzymatic Fragmentation Methods table
DNA Fragmentation
The majority of the current generation of next generation sequencing (NGS) platforms, including Illumina®, focus on short-read sequencing, which can be defined as sequencing library molecules between ~300 bp and 600 bp. DNA Fragmentation is, therefore, a necessary first step in most NGS library prep workflows. The two most common methods of DNA fragmentation for generating random double-stranded breaks without base bias are sonication/acoustic shearing, using instrumentation such as Covaris®, and enzyme-based fragmentation. The sound wave-based techniques are effective and widely used, but require specialized equipment that not all labs have access to, and can be challenging to scale. On the other hand, enzyme-based techniques are simpler and scalable, but have historically been trickier to fine tune.
NEBNext® Ultra™ II FS DNA Library Prep addresses the challenge of DNA fragmentation upstream of NGS library preparation with a unique fragmentation system that enables fragmentation, end repair, and d(A)-tailing with a single enzyme mix. The FS products offer tuneable enzymatic fragmentation based on incubation time, and independent of input amount. NEBNext Ultra II FS DNA Library Prep Kit is available with and without sample purification beads. For applications requiring enzymatic fragmentation, end repair, and d(A)-tailing without the rest of the library prep reagents, the NEBNext Ultra II FS DNA Module is available.
NEBNext UltraShear® stands apart from our other enzymatic fragmentation reagents in that it is compatible with methylation analysis workflows because it doesn’t erase or interrupt methylation marks. Its use is ideal for highly sensitive variant calling applications as it reduces artificial mutations, even in the most challenging samples. You can find more information on NEBNext UltraShear’s performance in our NEBNext UltraShear Data Supplement.
NEBNext UltraShear is incorporated in the NEBNext UltraShear FFPE DNA Library Prep Kit, which is specifically designed for processing FFPE DNA. This kit also contains the NEBNext FFPE DNA Repair Mix v2 to repair damage caused by deamination and oxidation during the fixation, storage, and extraction of FFPE DNA samples.
NEBNext dsDNA Fragmentase® is our original standalone enzyme-based reagent that fragments DNA in a time-dependent manner, but dsDNA Fragmentase is not part of the NEBNext Ultra II FS DNA reagents.
In contrast, newer NGS platforms offer the ability to sequence longer reads than ever before (e.g., Oxford Nanopore Technologies® (ONT), PacBio®) and, therefore, it is necessary to isolate intact high molecular weight DNA (HMW). Monarch® HMW Extraction Kits are available for a range of sample types, from cells and blood to tissue, and reproducibly purify HMW DNA, with tunable fragment length using a novel glass-bead-based approach.
Controlled fragmentation of HMW genomic DNA (gDNA) improves long-read sequencing performance and data yield. NEBNext UltraShear Long Read is a fast and tunable enzymatic gDNA fragmentation method that preserves native methylation. It offers ONT and PacBio users more usable bases, controlled fragment sizes and scalable throughput.
New England Biolabs (NEB) also offers companion reagents for use in ONT workflows; more information can be found here.
NEBNext Enzymatic DNA Fragmentation Methods
| APPLICATION |
Standard DNA Sequencing (10–200 ng) |
Standard DNA Sequencing (<10 ng and >200 ng) |
Library Prep for Methylation Analysis e.g. EM-seq™, Bisulfite Sequencing |
FFPE DNA Sequencing |
DNA Fragmentation Only |
Long Read DNA Sequencing | Direct Methylation Detection in Long Read DNA Sequencing |
|---|---|---|---|---|---|---|---|
| Sample Type |
Genomic DNA, intact DNA |
Genomic DNA, intact DNA |
Genomic DNA |
FFPE DNA | Genomic DNA, intact DNA |
HMW Genomic DNA, intact HMW gDNA |
HMW Genomic DNA, intact HMW gDNA |
| NEBNext UltraExpress® FS DNA Library Prep Kit (NEB #E3340) | +++ | – | – | – | – | – | – |
| NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® (NEB #E7805) | +++ | +++ | – | – | – | – | – |
| NEBNext UltraShear® (NEB #M7634) | ++ |
++ |
+++ | ++ |
+++ | – | – |
| NEBNext UltraShear® Long Read (NEB #E3430) | – | – | – | – | +++ | +++ | +++ |
| NEBNext UltraShear® FFPE DNA Library Prep Kit (NEB #E6655) | – | – | – | +++ | – | – | – |
| NEBNext® dsDNA Fragmentase® (NEB #M0348) | + | + | – | – | + | – | – |
| Table Legend: |
| +++ Optimal, recommended product for selected application |
| ++ Works well for selected applications |
| + Will perform selected application, but is not recommended |
| – Not recommended for this application |
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Improving Enzymatic DNA Fragmentation for Next Generation Sequencing Library Construction
Among the available options for DNA fragmentation, enzymatic fragmentation is demonstrably gentler on the sample, yielding less damage at any scale. The new NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina® includes a single step for enzymatic fragmentation, end repair, and dA tailing.
- Troubleshooting Guide for NEBNext® Ultra™ II FS DNA Library Prep Kit
- A Robust, Streamlined, Enzyme-based DNA Library Preparation Method Amenable to a Wide Range of DNA Inputs (2019)
- NEBNext® Ultra™II FS DNA: A Robust Enzyme-based DNA Library Preparation Method Compatible with Plant and Animal Samples (2019)
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