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URL: https://dx.doi.org/DOI:10.17504/protocols.io.6bthann

Authors: Marie-Ka Tilak, Rémi Allio, Frédéric Delsuc

Group: High molecular weight DNA extraction from all kingdoms

Summary: This protocol was developed to optimize DNA sequencing from mammalian roadkill tissue samples using Oxford Nanopore Technology (ONT). Roadkill tissues contain 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.

Affiliations: Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, EPHE, Université de Montpellier, Montpellier, France, Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, EPHE, Université de Montpellier, Montpellier, France, Institut des Sciences de l’Evolution de Montpellier (ISEM), CNRS, IRD, EPHE, Université de Montpellier, Montpellier, France

Version: 1

Publication Date: 2019

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