Thomas Couvreur: The long short cut to plant biodiversity
Thomas Couvreur from the Institut de Recherche pour le Developpement, opened his talk by discussing how global plant biodiversity is huge and many of the taxa present in the tropics are threatened by global climate change, for example deforestation. Other areas that Thomas and his colleagues are interested in are the evolutionary history of important crop species and the conservation and dynamics of tropical ecosystems. Due to this, Thomas stated that the need for rapid species identification is paramount to detected and analyse genomic data from novel, non-model organisms.
A number of challenges surround this aim, as plants typically have large complex genomes with large repeat regions and multiple copies of chromosomes. There is a distinct lack of reference genomes available and all these facts together make assembling these genomes a significant challenge.
In an attempt to overcome this, and provide rapid species identification, Thomas spoke of a method they had developed to target specific regions of the plants genetic material using short oligonucleotide probes hybridised to magnetic beads. This was performed, “in solution” in order to target chloroplast DNA, generally regarded as the best genetic barcode for plants. The reasoning behind using this approach was that it could increase multiplexing ability and read depth while reducing the analysis complexity. The benefits of using chloroplast sequences as a species ID tool is that it is a moderately long target, approximately 150, 000 bp, circular, haploid (so there is no phasing) and provides a good level of phylogenetic resolution for species typing. The downside is that it is in relatively low abundance compared with nuclear DNA and there are difficulties in de novo assembly.
Proposing some research questions, Thomas aimed to determine; how long were the sequences that were captured; what are the size ranges of these captured sequences; what was the coverage of the target; and do long reads improve plastome assembly.
Thomas and his team used the model grass species Oryza sativa to develop their sequence capture approach before moving on to use this method in 6 non non-model grasses and palms. At this point Thomas pointed out that the DNA from the model organism was fresh, however it is common for plant DNA samples to be shipped in a silica dried form. Therefore, a number of comparisons between silica dried and fresh DNA would be made. Summarising the method, Thomas said probes specific to plastid DNA were generated using fragmented products of long-range PCR products originating from a single plant species. These generated probes could be used to hybridise to chloroplast DNA and, via attached magnetic particles, pull down and enrich for chloroplast DNA.
When discussing the results, Thomas started by saying “It works!” and qualifying this with the fact that 70% of reads were of plastome in origin (5 fold over control) and the longest read was 26 kb with a median length of all reads being 4.2 kb. When examining coverage using this protocol on the model organism Oryza sativa average coverage was 364 x with 100 % coverage at 10 x and 99.9996 % with a coverage of 50 x. In the non-model organisms an increase from 15 % to 98 % of sequences were of plastid origin when compared with controls and fold coverage increased from 12 – 156 X. Fragment length distributions in the non-model organisms ranged from a median read length of 3.4 kb to 4.6 kb using fresh DNA extracts but were shorter when silica gel preserved DNA was used. These non-model organisms had an average coverage of 500 x although some gaps were present but 96 % was covered to 10 x depth and 91 % to 50 x. As a result, the fresh DNA samples produced assembled plastids in two contigs for each fresh dna samples while Silica gel dried samples produced 10 – 17 contigs.
Concluding, Thomas summed up the benefits of the sequence capture protocol they developed as capturing long fragments from plats was possible and that although the generated assemblies were good, manual finishing would significantly improve these.