Isolation of High Molecular Weight DNA from the Model Beetle <i>Tribolium</i> for Nanopore Sequencing

• Main Authors: Marin Volarić, Damira Veseljak, Brankica Mravinac, Nevenka Meštrović, Evelin Despot-Slade
• Format: Article
• Language: English
• Published: MDPI AG 2021-07-01
• Series: Genes
• Subjects: beetle; high molecular weight DNA; Nanopore sequencing
• Online Access: https://www.mdpi.com/2073-4425/12/8/1114

The long-read Nanopore sequencing has been recently applied for assembly of complex genomes and analysis of linear genome organization. The most critical factor for successful long-read sequencing is extraction of high molecular weight (HMW) DNA of sufficient purity and quantity. The challenges associated with input DNA quality are further amplified when working with extremely small insects with hard exoskeletons. Here, we optimized the isolation of HMW DNA from the model beetle <i>Tribolium</i> and tested for use in Nanopore sequencing. We succeeded in overcoming all the difficulties in HMW handling and library preparation that were encountered when using published protocols and commercial kits. Isolation of nuclei and subsequent purification of DNA on an anion-exchange chromatography column resulted in genomic HMW DNA that was efficiently relaxed, of optimal quality and in sufficient quantity for Nanopore MinION sequencing. DNA shearing increased average N50 read values up to 26 kb and allowed us to use a single flow cell in multiple library loads for a total output of more than 13 Gb. Although our focus was on <i>T. castaneum</i> and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.