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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bhftj3nn

Authors: Roland Wouters, Sam Mugford, Roberto Biello, Darren Heavens, Saskia Hogenhout

Group: High molecular weight DNA extraction from all kingdoms

Summary: Chromosome-scale genome assembly usually requires the use of long-read sequencing technologies such as PacBio or ONT MinION. However, the development of suitable DNA extraction methods that can yield high molecular weight DNA suitable these platforms can be problematic and time-consuming. We found that some traditional DNA extraction methods that are suitable for short-read sequencing platforms did not produce satisfactory results using long-read sequencing technologies with aphids or other sap-feeding hemipteran insects. We hypothesised that plant-derived components of the aphid’s diet might inhibit the sequencing chemistry. Here we present a method suitable for extraction of high-molecular DNA for long-read sequencing of aphids and other sap-feeding insects. We employed the Illustra Nucleon Phytopure DNA extraction kit designed to remove plant-specific contaminants. We were able to consistently recover very high molecular weight DNA that was compatible with library preparation and sequencing by PacBio or ONT MinION. The protocol presented here describes the use of this plant-DNA extraction kit optimised to obtain high molecular weight DNA from insect samples.We have used this method across a range of aphid species, leafhoppers and froghoppers (spittlebugs). It has enabled sequencing and - in most cases -chromosome-level assembly of genomes from the green peach aphid Myzus persicae (1), the pea aphid Acyrthosiphon pisum (1), the woolly apple aphid, Eriosoma lanigerum (2), and the common meadow spittlebug Philaenus spumarius (3). 1. Chromosome-scale genome assemblies of aphids reveal extensively rearranged autosomes and long-term conservation of the X chromosomeThomas C.Mathers, Roland H. M.Wouters, Sam T.Mugford, DavidSwarbreck, CockVan Oosterhout, Saskia A.HogenhoutbioRxiv 2020.03.24.006411; doi: https://doi.org/10.1101/2020.03.24.006411 2. A chromosome-level genome assembly of the woolly apple aphid, Eriosoma lanigerum (Hausmann) (Hemiptera: Aphididae)RobertoBiello, ArchanaSingh, Cindayniah J.Godfrey, FelicidadFernández Fernández, Sam T.Mugford, GlenPowell, Saskia A.Hogenhout, Thomas C.MathersbioRxiv 2020.05.29.121947; doi: https://doi.org/10.1101/2020.05.29.121947 3. Draft genome assembly version 1 of the meadow spittlebug Philaenus spumarius (Linnaeus, 1758) (Hemiptera, Aphrophoridae) (Version 1) [Data set]. Roberto Biello, Thomas C. Mathers, Sam T. Mugford, Qun Liu, Ana S. B. Rodrigues, Ana Carina Neto, Maria Teresa Rebelo; Octávio S. Paulo; Sofia G. Seabra; Saskia A. Hogenhout. (2020). Zenodo. http://doi.org/10.5281/zenodo.3368385

Affiliations: John Innes Centre, Norwich, UK, John Innes Centre, Norwich, UK, John Innes Centre, Norwich, UK, Earlham Institute (EI), Norwich, UK, John Innes Centre, Norwich, UK

Version: 1

Publication Date: 2020

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