Introduction
Ligation Sequencing Kit protocols contain no designated fragmentation step. However, fragmentation of genomic DNA has been shown in some cases to provide benefits in:
- Reduction in library input requirements
- Increasing read length N50
- Reduction in pore blocking
This protocol contains information regarding the use of Megaruptor® 3 (Diagenode) for fragmenting gDNA for subsequent library preparation using the Ligation Sequencing Kit. Size selection of gDNA (for example, using the protocol ‘Size selection of HMW DNA by semi-selective DNA precipitation’) can be performed before Megaruptor® 3 shearing to help remove the shortest fragments.
Materials
- 2000 ng gDNA in 100 μl nuclease-free water
- Diagenode Megaruptor® 3
- Megaruptor 3 Shearing Kit : Cat. No. E07010003
- Nuclease-free water
- DNA QC equipment such as the Agilent FEMTO Pulse
- Microfuge
Method
- Optional step: Perform a size selection on your DNA sample using the Size selection of HMW DNA by semi-selective DNA precipitation protocol.
- Transfer 2000 ng of genomic DNA into a 0.5 ml sample tube supplied in the Megaruptor 3 shearing kit, and adjust the volume to 100 μl with nuclease-free water. Mix the DNA thoroughly by pipetting the sample. Spin down briefly in a microfuge.
- Transfer the sample to the Megaruptor 3, enter the shearing parameters into the instrument, and start the run.
- When the shearing step is complete, remove the fragmented DNA from the instrument.
- Analyse 1 μl of the fragmented DNA for fragment size (for example using the Agilent FEMTO Pulse).
- Use 1 µg of fragmented DNA in Ligation Sequencing Kit library preparation.
Results
Figure 1. Fragment length profile after Megaruptor 3 shearing. Input DNA was fragmented using a variety of shearing speeds and the resulting DNA was analysed by FEMTO Pulse. A shearing speed of 3 appears to have had little impact on the fragment length profile, suggesting unsuccessful fragmentation. As the shearing speed is increased, the sample becomes progressively more fragmented.
Change log
Version | Change |
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v1, September 2019 | Initial protocol publication |