Abstract Detail

Tropical Biology

Ortiz, Edgardo M. [1], Hoewener, Alina [1], Andriamiharisoa, Minosoa [2], Shigita, Gentaro [1], Radanielina, Tendro [2], Schaefer, Hanno [1].

Assessing Cucurbitaceae diversity in Madagascar using Nanopore-based field genomics.

Using sequence capture data, we built a genomic database for Cucurbitaceae, which can now be used for the identification of plant material during field work. We rely on portable laboratory equipment and Nanopore’s sequencing device MinION. Our laboratory protocol and analysis pipeline allow the genetic analysis and identification of fresh leaf samples which helps to steer collection efforts and ecological data collection almost in real time. We tested our approach in Madagascar, one of the most endangered biodiversity hotspots worldwide, where we expect a large number of overlooked cucurbit taxa. We chose three of the most informative orthologs of the Angiosperm353 set and the ITS and ETS regions. These five regions are simultaneously amplified in a multiplexed PCR reaction using all primers combined in an optimized concentration for each pair. Barcoded Nanopore sequencing libraries are prepared from the resulting amplicons and sequenced on a Flongle flowcell with the MinION device. Our analysis pipeline ‘NanoAmp’, starts by basecalling the data using a GPU with Nanopore’s ‘Guppy’ software. Raw sequencing reads are then quality-filtered using ‘bbduk’ from ‘BBTools’, and cleaned reads are used for de novo assembly with amplicon-optimized settings in ‘Canu’. Since long reads are able to potentially separate alleles for each marker, we then perform variant calling and haplotyping using ‘Longshot’. Finally, haplotyped assemblies are polished using Nanopore’s ‘Medaka’ software. In order to sort the assembled contigs by marker and align them across samples we use our pipeline ‘Captus’. The resulting alignments are integrated into an alignment containing all the samples of our reference database and a phylogeny is inferred using RAxML or IQ-TREE. After interpretation of the phylogenetic results, the new samples are assigned to species, while simultaneously growing our reference database. We collected 16 taxa during the first field trip and our database comprises now 67% of the 70 species currently known for Madagascar. A major result is that given enough sequencing depth per marker, the final assemblies show accuracies comparable to Sanger sequencing which allows correct phylogenetic placement of the samples, while being able to additionally distinguish alleles for the markers selected.

1 - Technical University of Munich, Life Science Systems, Plant Biodiversity Research, Emil-Ramann Strasse 2, Freising, D-85354, Germany
2 - University of Antananarivo (UA), Department of Plant Biology and Ecology (DPBE), Faculty of Science, Madagascar

amplicon assembly
field genomics
molecular identification
multiplexed PCR

Presentation Type: Oral Paper
Number: TP1001
Abstract ID:412
Candidate for Awards:None

Copyright © 2000-2022, Botanical Society of America. All rights reserved