Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing

Volarić, Marin; Veseljak, Damira; Mravinac, Brankica; Meštrović, Nevenka; Despot-Slade, Evelin (2021) Isolation of High Molecular Weight DNA from the Model Beetle Tribolium for Nanopore Sequencing. Genes, 12 (8). ISSN 2073-4425

Preview

PDF - Published Version - article
Available under License Creative Commons Attribution.
Download (6MB) | Preview

Official URL: https://www.mdpi.com/2073-4425/12/8/1114

Abstract

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 Tribolium 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 T. castaneum and closely related species, we expect that this protocol, with appropriate modifications, could be extended to other insects, particularly beetles.

Item Type:

Article

Uncontrolled Keywords:

beetle ; high molecular weight DNA ; Nanopore sequencing

Subjects:

NATURAL SCIENCES > Biology

Divisions:

Division of Molecular Biology

Projects:

Project title	Project leader	Project code	Project type
(Epi)genomika i transkriptomika eukromatinskih satelitnih DNA u embriogenezi i razvoju	Meštrović Radan, Nevenka	IP-2019-04-4910	HRZZ
Rasvjetljavanje evolucije satelitnih DNA visokoprotočnim analizama satelitoma srodnih vrsta	Mravinac, Brankica	IP-2019-04-5522	HRZZ

Depositing User:

Brankica Mravinac

Date Deposited:

25 Aug 2021 09:03

URI:

http://fulir.irb.hr/id/eprint/6540

DOI:

10.3390/genes12081114