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Vania Costa - The fever tree: extracting and preparing the DNA of Cinchona pubescens

London Calling 2019

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Vania Costa (Applications group, Oxford Nanopore Technologies) began her talk by introducing Cinchona, a plant that has saved countless lives over the past several hundred years. Known as the "fever tree", Cinchona species are native to the Cloud forests of the Eastern Andes, and are now distributed through Africa, Southeast Asia and Australia. Cinchona species are a source of the anti-malarial alkaloid quinine, which for centuries was the only effective anti-malarial and remains of high importance in malaria treatment to this day.

Oxford Nanopore are working in collaboration with Kew Gardens to sequence Cinchona pubescens, cultivated at Kew. The project aims to shed light on how Cinchona species are related, and which genes are involved in the quinine biosynthetic pathway, to help determine which species makes the best source of quinine - and to potentially identify other effective anti-malarial alkaloids. Vania outlined the typical workflow of a sequencing project, from sample to extraction, QC, sequencing and finally data analysis; she then discussed the initial considerations for Cinchona that went into selecting a suitable extraction method for the project. The genome has been estimated to be between 600 Mb and 1.1 Gb, and is rich in alkaloids including quinine, which unfortunately can inhibit DNA extraction.

Vania collected leaves from a C. pubescens tree cultivated at Kew Gardens, then selected seven plant DNA extraction methods for testing. Noting that different extraction methods often provide a different balance between read length and yield, Vania selected Macherey-Nagel Nucleospin Plant II for its high yield and a method combining Calson buffer and QIAGEN Genomic-tip 500/G to aim for maximum fragment lengths. Vania talked through the protocol for the former method; the libraries prepared in this way were sequenced on MinION, GridION and PromethION and produced good yields on each, with a PromethION Flow Cell producing over 60 Gb data in 24 hours. Samples extracted with the latter method were size selected to further improve read length distribution: Vania showed how the Circulomics Short Read Eliminator Kit significantly improves read length distribution. Though yield was slightly lower in sequencing for these samples (over 50 Gb on the PromethION in 24 hours), the read N50 was over 48 kb: up from 28 kb without size selection).

Vania concluded by stressing the importance of selecting a good extraction method, as the baseline for good results in sequencing - "you cannot sequence what you don't have" and of selecting an extraction method and post-extraction prep that fits with the goals of the experiment. For more information, Vania highlighted the Protocol Builder, available on the Community, an interactive tool that starts with the goals of an experiment and works backwards, outputting a bespoke extraction-to-analysis pipeline. The Extraction Methods section of the Community also features further information on extraction methods for a wide range of samples, together with protocols for size selection, guidance on contaminants and other tips and tricks.

The optimised protocols Vania developed were used to extract and prepare the C. pubescens libraries which will be sequenced live on the PromethION in the Live Lounge throughout London Calling; the data generated will then be used by Kew Gardens to help research the plant's origins and its invaluable anti-malarials.

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