NGS Success - DNA Shearing
View next generation sequencing (NGS) tips for DNA shearing:
- Check shear settings for make/model of ultrasonicator as shearing settings may vary.
- Maintain proper water levels for that machine.
- Make sure there are no bubbles in assay tube.
- Set up reactions on ice to ensure consistency for all samples. The enzyme has a low activation temperature and will start prematurely at room temperature.
- Timing of EDTA addition to stop reaction is important, adjust incubation time as necessary but ensure reaction stopped consistently for all samples.
- Good to consider: the use of fragmentase in lieu of physical fragmentation can have a slightly adverse effect on QC metrics (such as increased %duplication and mean target coverage).
- When using fragmentase for shearing, make sure DNA is diluted or eluted in low EDTA solution during extraction as Mg2+ will inhibit the enzyme’s activity.
Figure 1: Comparison of mechanical and enzymatic fragmentation methods using Agilent High Sensitivity D1000 ScreenTape assay. Size distribution should be with a peak between 150 to 250 bp (+/– 10%). Samples treated with fragmentase display a broad shoulder indicative of a wide-range of fragment sizes = Blue Trace. Samples sheared by Covaris (or similar) will have a narrow peak = Orange trace.
Figure 2. Comparison of Enzymetic fragmentation incubation times using Agilent High Sensitivity D1000 ScreenTape. Graphs show increasing the fragmentation time results in a gradually decreasing peak size corresponding to a reduced yield of DNA.