While the preparation of intact high molecular weight DNA and prevention of shearing throughout most workflow steps is important, DNA fragmentation is a necessary step in sample prep for most sequencing platforms. For example, short read sequencing platforms generally rely on fragments of ~300-600 bp), while long read sequencing platforms are compatible with fragments many kb in length.
It is essential to choose a fragmentation method that reliably generates appropriately sized fragments without introducing bias as to the cut sites or base composition of the fragments. There are several options for the DNA fragmentation step, each with its own unique profile of benefits and drawbacks, and the selection of a fragmentation method should be carefully weighed. Methods used include:
· Enzyme-based treatments: These fragment DNA by simultaneously cleaving both strands, or by generating nicks on each strand of dsDNA to produce dsDNA breaks. This method is highly flexible and can be used for generation of fragments from low bp to many kb in length.
· Acoustic shearing: Short-wavelength, high-frequency acoustic energy is focused on the DNA sample, physically disrupting the DNA molecule, without requiring temperature changes. This method is used for generation of fragments from low hundreds of bp to many kb in length.
· Sonication: Specialized sonicators subject DNA to unfocused, longer-wavelength acoustic energy; sonicators require a cooling period between sonication bursts. This method is used for generation of fragments many kb in length.
· Centrifugal shearing: DNA can be sheared by the use of centrifugal force to move DNA through a hole of a specific size; the rate of centrifugation determines the degree of DNA fragmentation. This method is used for generation of fragments many kb in length.
· Point-sink shearing: A syringe pump generates hydrodynamic shear forces within a tube; the size of the constriction and the flow rate of the liquid determine the DNA fragment size. This method is used for generation of fragments many kb in length.
· Needle shearing: The lowest-tech option relies on shearing forces created by passing DNA through a small gauge needle. This method is used for generation of fragments tens of kb in length.
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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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.