From the very basic problems of how to break up different kinds of tissue in order to extract DNA, to working with the "pre-fragmented" DNA that can be found in historic specimens, working in a Botanical Garden laboratory is full of challenges.
Developing best practises for field collection of plant material
The quality and quantity of DNA recovered from stored plant material is becoming more important as DNA sequencing technologies change from relatively simple Sanger sequencing to high-throughput sequencing technologies.
Whether it is the need for large quantities of pure DNA from our living collections for Genome assembly projects, including telomere-to-telomere sequencing, or minute amounts of degraded DNA from our historical collections for a targeted enrichment approach, a successful DNA extraction protocol is a crucial first step on the road to generating robust genetic data.
Extracting the most from our collections
We are constantly developing and refining standard operating protocols (SOPs) for the enormous diversity of taxa and tissue types we research at RBGE – from testing methods of tissue disruption on fungal spores to dealing with polysaccharides that co-extract with DNA.
While the gold standard for sample tissue storage is cryopreservation in liquid nitrogen, this is not a viable option for most field collections or for small sized or resource-poor institutes. We are currently investigating the effects of different specimen preservation techniques on the quantity and quality of DNA extractions, with a long-term experiment that includes sample collection into liquid nitrogen, buffered solutions and silica gel.
Plants and fungi contain a wide range of secondary chemicals, which can interfere with downstream sequencing applications. The RBGE laboratories invest in the development of SOPs for high-molecular weight (HMW) and high-quality DNA extraction methods, a basic pre-requisite for long-read sequencing success, particularly using Oxford Nanopore Technologies (ONT) and Pacific Biosciences (PacBio).
See our high molecular weight DNA extraction method for PacBio HiFi sequencing here
The specimens stored in herbaria contain a wealth of information about plant diversity and distributions; however, assessing the genetic component of this diversity can be hampered by the degraded quality of DNA after many years of storage. The successful application of advanced sequencing techniques to historical or processed plant material has revolutionised how we use our preserved collections in molecular research, and we have developed SOPs for extractions from historic or very valuable material, to maximise sequencing success while minimising environmental and cross-sample contamination.
Read about the Limits of Hyb-Seq for Herbarium Specimens: Impact of Preservation Techniques
Focusing on our core research taxa, we are developing SOPs for library preparation and targeted enrichment, or “hybrid capture”, techniques to retrieve large scale genetic data from both our herbarium and silica gel-preserved collections. Not all commercial library prep kits work equally well on plant DNA, so this development involves testing a range of different methods and kits so we can maximise the genetic data we obtain from our samples.
Retrieval of hundreds of nuclear loci from herbarium specimens
We have also developed SOPs for ONT-based methods for multiplexed amplicon sequencing, as an alternative to Sanger sequencing.