DNA FragmentationThe 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 soundwave-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 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.
NEBNext Fast DNA Fragmentation & Library Prep Set for Ion Torrent™ includes enzymatic fragmentation in a streamlined library prep workflow.
In contrast, newer NGS platforms offer the ability to sequence longer reads than ever before (e.g., Oxford Nanopore Technologies®) and, therefore, it is necessary to isolate intact high molecular weight DNA (HMW). The Monarch® HMW Extraction Kits are available for a range of sample types, from cells and blood to tissue, and reproducibly purify HMW DNA, with tuneable fragment length using a novel glass-bead-based approach. New England Biolabs (NEB) also offers companion reagents for use in Oxford Nanopore Technologies workflows; more information can be found here.
RNA FragmentationLike DNA sequencing, RNA sequencing typically requires inputs of a certain size/length and quality. RNA fragmentation can be achieved with the same basic methodologies as DNA fragmentation: sonication/acoustic shearing and enzyme-based reagents.
The NEBNext® Magnesium RNA Fragmentation Module enables the fragmentation of RNA using enzymes, heat alone, or exposure to divalent cations at elevated temperatures. The fragmentation is highly efficient and tuneable by simply incubating with Magnesium ions at 94˚C.
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)
Products and content are covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB). The use of trademark symbols does not necessarily indicate that the name is trademarked in the country where it is being read; it indicates where the content was originally developed. The use of this product may require the buyer to obtain additional third-party intellectual property rights for certain applications. For more information, please email email@example.com.
This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
This video describes some things to take into consideration when deciding on a size range for your DNA fragmentation.
Get a quick overview of NGS library preparation from an NEB scientist.