We are happy to share our optimized protocol for sequencing long reads from mammalian roadkill tissues with the Oxford Nanopore Technology (ONT). This protocol was specifically developed to optimize DNA extraction and sequencing roadkill tissues containing necrotized cells and impurities that result in DNA degradation. First, we observed that DNA preservation was generally better in RNAlater than in 95% EtOH preserved tissues. Second, we found that physically removing necrotized and epidermal cells before DNA extraction resulted in better DNA quality and purity. Finally, adjusting the ratio of AMPure beads to 0.4x at the DNA purification step permitted optimizing size selection for subsequent ONT library construction. These optimization steps allowed to significantly increase both read length and yield per flow cell for roadkill samples sequencing on the ONT MinION device. Try it to make the most out of roadkill!
Allio R., Tilak M.-K., Delsuc F. (2020). An optimized protocol for sequencing mammalian roadkill tissues with Oxford Nanopore Technology (ONT). protocols.io. doi:10.17504/protocols.io.beixjcfn.
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