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URL: https://dx.doi.org/DOI:10.17504/protocols.io.5isg4ee

Authors: Ashley Jones, Ramawatar Nagar, Anna Sharp, Benjamin Schwessinger

Group: High molecular weight DNA extraction from all kingdoms

Summary: Extracting pure high-molecular weight DNA from some fungal species is difficult due to the presence of polysaccharides and potentially other compounds which biochemically mimic DNA or interfere with the DNA extraction process. Such compounds can co-elute with DNA in many extraction methods, being difficult to separate fom the DNA. Although the contaminant may not be detected by spectrophotometers or fluorometric devices, it substantially interferes with long-read DNA sequencing, such as Oxford Nanopore Technologies. To partially resolve this, a protocol is presented with some updates to current strategies and incorporates a gel purification with a Pippin Prep (Sage Science). Using this protocol, we have been successfully sequencing the wheat stripe rust Puccinia striiformis and leaf rust Puccinia triticina with a MinION (Oxford Nanopore Technologies). Sequencing yields have surpassed 4 gigabases with an N50 of approximately 30 kb. To increase sequencing output, more work is needed to identify and remove the elusive contaminants.

Affiliations: Australian National University, Australian National University, Australian National University, Australian National University

Version: 2

Publication Date: 2019

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