Tips include why you should set up reactions on ice and advice for the use of fragmentase in lieu of physical fragmentation.
Check shear settings for make/model of ultrasonicator as shearing settings may vary.
Pete Gray
Array and NGS Field Application Specialist, Europe
Maintain proper water levels for ultrasonicator.
Huiyan Jin
NGS Field Application Scientist, North America
Make sure there are no bubbles in assay tube.
Serena Pace
NGS Field Application Scientist, North America
Ensure all of the sheared sample is removed from the assay tube to maximise recovery.
Kicki Bergefall
FAS Manager - Europe
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.
Serena Pace
NGS Field Application Scientist, North America
Timing of EDTA addition to stop reaction is important, adjust incubation time as necessary but ensure reaction stopped consistently for all samples.
Pete Gray
Array and NGS Field Application Specialist, Europe
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).
Huiyan Jin
NGS Field Application Scientist, North America
When using fragmentase for shearing, make sure DNA is diluted or eluted in low EDTA solution during extraction as a reduced Mg2+ concentration will inhibit the enzyme’s activity.
Kicki Bergefall
FAS Manager – Europe
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.
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