NGS Success - DNA Shearing

View next generation sequencing (NGS) tips for DNA shearing:

Mechanical

  • 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.

Enzymatic

  • 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.

Fig 1: Comparison of Mechanical and Enzymatic fragmentation methods 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.

Fig. 2. Comparison of Enzymetic fragmentation incubation times 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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