With a wealth of experience in genomics and plant biology, Ashleigh Lister is applying her expertise to single-cell genomics - pioneering detailed analysis of pollen grains that can give an unprecedented boost to crop breeders.
With a wealth of experience in genomics and plant biology, Ashleigh Lister is applying her expertise to single-cell genomics - pioneering detailed analysis of pollen grains that can give an unprecedented boost to crop breeders.
Though bitterly cold outside, alone in the climate-controlled greenhouses outside the Earlham Institute Ashleigh Lister has spent the winter months intricately dissecting the anthers of wheat plants. A leader in her field, she is applying the transformative potential of single-cell genomics to wheat breeding for the very first time.
What she discovers will advance our understanding of recombination in hybrid wheat, and allow breeders to generate new varieties more quickly and with greater success.
I want to bring my experience of working on wheat into the single cell world. I don't think the plant sciences have really gone to single-cell resolution, not in wheat anyway.
I’m currently looking at microspores, the progenitor cells of pollen, taken from the anthers. I isolate them through our cell sorter, then perform G&T sequencing [we’ll explain more later]. I'm trying to do this on parental lines, then hybrid lines, and see how that changes the recombination switches and the different mapping of genes.
Say if I was to look at a hybrid: I've got Chinese spring, and I've got Paragon, the two cultivars that are the parents of that hybrid. I'm looking for a switch in the genomic sequence from one into the other, which has occurred during meiosis. I'm also looking for recombination hotspots, and where they lie along a chromosome.
It’s something that breeders are really trying to explore a little bit more because you tend to get a dry zone within a chromosome that never gets introduced into a new cultivar. The main thing is to get coverage across the genome, and we’re then looking at the RNA to see if we can spot anything useful.
All the breeders want to be able to do this but don't have the specific expertise and equipment, such as flow sorters or the genomics facilities. Here at EI, we’ve got it all under one roof.
It's not the drink, as cool as that sounds. It stands for genome and transcriptome sequencing and can be performed on the same single cell.
We sort and process around 100 cells at the same time using robots, which makes things both easier and standardised. Then, the first part of every RNA sequencing (RNA-seq) pipeline is called a pull down - we use beads which bind to the poly-A tail to pull out all of the mRNA transcripts that are present in any one cell at the time point you've sorted it and lysed it. Everything that's washed off of the bead and not bound to it is DNA plus other components of the cell.
We clean that up separately and then process to two plates, one for RNA-seq and one for DNA-seq. There are other things that can work alongside it, such as DNA methylation sequencing. There's more in development but that that's the most common method we currently use.
Image below: Wheat microspores stained with a dye which picks up on the DNA in the cell.
I can receive a sample and have it sequenced within the same week, potentially. That's all because everything is in the same building. We have the expertise for the whole pipeline, really.
At EI we tend to work on things that don't have a protocol yet. We do lots of technology development, which is really fun.
We have an unbelievable suite of fancy instruments to do that - it’s outstanding. People approach us to look at really weird and wonderful things, which means that we have several different instruments for single-cell isolation that are all excellent for different applications. We essentially have a cell-sorting suite, which is quite unique to the Institute and an asset to the region.
On top of that, we have robotics and automation platforms - and we are lucky to be working directly with the sequencing team in Genomics Pipelines. That really helps because I've worked in that group, so I am constantly talking to them and asking them for advice and vice versa.
I can receive a sample and have it sequenced within the same week, potentially. That's all because everything is in the same building. We have the expertise for the whole pipeline, really.
The other thing is that it’s very unusual to have a boss [Iain Macaulay] who knows how to run all of the instruments, and how to do this stuff in the lab at the ground level. That's very, very useful. He knows that things sometimes go awry, and he has advice and backup plans as well. That's also very good for setting up collaborations, as he can be in those meetings and understand projects from the outset.
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Many might relate to the feeling that, when you send a sample for sequencing, it goes away to this magical black box, something happens and you get data out of the other end. Nobody really understands quite what happens unless you're doing it.
So, then I got to do the thing that happens in the black box and it was incredibly useful for everything I do in my work now. Genomics Pipelines are very good like that. They get you set up with experience of every stage that the sample would go through. I would appreciate quality control - what a good sample and a bad sample look like, and how that would then affect the next stage of library construction.
Then there's the sequencing side at the end, which is obviously vital, and is extremely interesting. It's nice to have appreciated what Tom [Barker], Naomi [Irish] and Vanda [Knitlhoffer] do. It's quite a job, and quite a responsibility to keep those instruments going all the time.
I can't ever do biology in bulk now. Each cell is the foundation and the building block of a tissue and an organism, and then that species, and therefore it's so important to understand how that cell type behaves. It can completely flip your functional understanding on its head.
Laura [Mincarelli] and Anita [Scoones], in fact most of my group, work on blood development - and that has been worked on forever. It’s probably the most studied of any cell type at any point - it's certainly been worked on a single cell level for a very long time - and yet they're still discovering new things about it, especially at the single cell level.
The single-cell field has new things going on all the time. We can use different platforms, such as long read platforms, which change what you pick up on in that cell. You can apply RNA or DNA to it, or you could look at the methylation. You can look at ATAC (Assay for Transposase-Accessible Chromatin) and nuclear zone associations - gene regulation, where those genes are sat in the chromosome, the organisation of it all. And all of those things can be read out in different algorithms.
It's the advancement of single-cell genomics all the time, and the understanding that comes with it.
