Novel tools for reducing bias in Next Generation Sequencing of small RNAs

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Published on: April 15, 2014

Tamas Dalmay, Professor of RNA Biology at the University of East Anglia (Norwich), has developed a robust, simple method of profiling small RNAs using next generation sequencing. Here he explains his novel HD adapters and why they are more reliable than existing commercial adapters. 

Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).
Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).

Small RNAs (sRNAs) are key regulators of gene expression, and accurate representation of sRNA in sequencing experiments is critical to the interpretation of biological data. Next generation sequencing (NGS) is now the gold standard for profiling and discovering new sRNAs, so it is essential that the tools and protocols used in NGS generate accurate, reliable sequence data.

RNA ligases are essential in creating cDNA libraries prior to NGS sequencing. However, a number of recent publications reported that RNA ligases used in cDNA preparation actually mediate sequence specific ligation, so NGS approaches using these RNA ligases do not represent all sRNA present in biological samples. These publications highlighted the limitations associated with RNA ligases, questioning the reliability of currently widely used NGS approaches and the data generated from them.

Sequence specific ligation occurs because the ligases preferentially ligate ends that are more likely to be close to each other. This means that sRNAs that can efficiently anneal to the adapters have a higher chance of being ligated (Jayaprakash et al. 2011, Hafner et al. 2011 and Sorefan et al. 2012).

While identifying that cloning bias in sRNA libraries is RNA ligase dependent, our group at the School of Biological Sciences, University of East Anglia (Norwich), developed a novel, simple, robust solution to overcome this problem (Sorefan et al. 2012).

We developed a set of adapters (High Definition or HD adapters) that contain degenerated nucleotides, meaning they are a pool of many sequences instead of one fixed sequence. Consequently, many different sRNAs can form a stable duplex with them, leading to better coverage and more quantitative libraries. We have shown that using the HD adapters: (more…)

Photosynthesis for fresh water

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Published on: February 10, 2014

Annegret Honsbein is a post-doc in Anna Amtmann‘s lab at the University of Glasgow. As she explains in this guest post, she is working on an EPSRC project that hopes to harness the power of photosynthesis to desalinate sea water. 

Water covers more than 70% of Earth’s surface but less than 2% of it is available as freshwater. Many of the driest regions of our planet are close to the sea but irrigating fields with seawater – even if diluted – leads to build-up of salt in the soil to levels toxic to all common food crops. Current desalination technologies, such as membrane-based reverse osmosis, are successfully used in large-scale desalination plants, but they are expensive and energy inefficient.

desalinationOur multi-disciplinary EPSRC-funded project takes a synthetic biology approach to the development of an innovative desalination technology based on biological processes. We are a team of biologists and engineers from the Universities of Glasgow, Sheffield, Newcastle, Robert Gordon University at Aberdeen and Imperial College London, led by Dr. Anna Amtmann from Glasgow University.

Our idea is solar energy-fuelled desalination – with a twist. Instead of using solar panels we intend to let photosynthetic microorganisms desalinate the sea water. Cyanobacteria are ideal candidates, and we are currently working with two strains that are naturally able to adapt to a wide range of salt concentrations from fresh to sea water.

In principle, salt is toxic to all living cells, which is why most living systems have developed means to actively export sodium. In some cyanobacteria species that grow to very high densities, this ability means they actually form a low-salt reservoir within their saline environment.

We intend to use this low-salt reservoir as ion exchanger to extract the salt from the surrounding seawater. We aim to engineer cyanobacteria so we can switch off the endogenous salt export mechanism towards the end of their growth cycle, and activate a synthetic intracellular sodium accumulation unit. This synthetic unit will be assembled from membrane transport proteins evolved by different organisms to import sodium and chloride ions.

Our team’s engineers are developing techniques to manipulate the surface properties of the cyanobacteria and effectively separate the ‘salty’ cells from the desalinated water before they die, preventing release of the accumulated salt back into the ‘fresh’ water.

The final stage of the project will be to build a model version of the actual plant that could house our photosynthesis-driven bio-desalination process.

This work is published in: Jaime M. Amezaga, Anna Amtmann, Catherine A. Biggs, Tom Bond, Catherine J. Gandy, Annegret Honsbein, Esther Karunakaran, Linda Lawton, Mary Ann Madsen, Konstantinos Minas and Michael R. Templeton (2014) Biodesalination: A Case Study for Applications of Photosynthetic Bacteria in Water Treatment. Plant Physiology 164: 1661-1676; doi: http:/​/​dx.​doi.​org/​10.​1104/​pp.​113.​233973.

Image c/o Annegret Honsbein.

How do plants remember winter?

Martin Howard is a Professor at the John Innes Centre, one of a small cluster of research institutes in Norwich. In the fourth of our Celebrating Basic Plant Science series, he explains how he uses mathematical modelling to understand how plants remember winter cold and respond to it throughout the year. 

How do plants ‘know’ the correct time to flower? Getting this timing right is vital for reproductive success; flowering in the middle of winter is unlikely to be optimal! Many factors are integrated together to make this critical decision, including the day length.

We have been studying one aspect of this question: How the plant Arabidopsis thaliana perceives and then remembers exposure to winter cold. This fundamental mechanism ensures that flowering doesn’t occur until winter has passed. Interestingly, this memory is quantitative – a longer winter means flowering is faster once it starts (see the image below).  This process is a very nice example of what’s called an epigenetic phenomenon, as the plants store information about winter cold exposure even after the environmental stimulus (cold) has been removed.

So how is this information about cold stored? In Arabidopsis, this is centred on a gene called FLC (Flowering Locus C). When the plant is cold, the FLC gene is turned off. The products of this gene prevent flowering, so turning it off actually stimulates the plant to flower. Over recent years, we have learned a great deal about the operation of FLC and associated genes through genetics and biochemistry, in large part through the work of my experimental collaborator, Caroline Dean. However, despite all this knowledge it was still not clear overall how the epigenetic memory system worked. This was partly due to feedback among the different components, which made arriving at an intuitive understanding a very difficult task. For these reasons, we began to model the dynamics of FLC mathematically in the hope of making sense of these interactions and, we hoped, revealing some underlying simplicity in how the system operated.

Mathematical modelling turned out to be very informative and suggested that FLC gene silencing occurred in an all or nothing fashion inside each cell. (more…)

What makes one species different from another?

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Published on: December 10, 2013

In the third of our series of blog posts Celebrating Basic Plant Science, Catherine Kidner from Edinburgh’s Royal Botanic Garden explains how she pinpoints what makes a species a species. This sheds light onto what drove that species’ evolution. Catherine spoke about her research at a conference earlier in the year – you can see her talk in this video

Catherine Kidner uses genomic data to understand why Begonia like these have evolved into different species

There are likely to be about 9 million species on Earth (not counting bacteria). Each species has traits which define it and allow it to thrive in its niche. My research group at the Royal Botanic Garden in Edinburgh is trying to understand the diversity around us, so we need to know what these traits are and how they contribute to the success, or otherwise, of the species.

There are difficulties with this approach to understanding diversity. For a start, traits we think are important may not be that important to the species concerned. Also, some traits critical to a species’ success might be difficult to measure, for example phosphate uptake by roots; or be seen only on very specific occasions, like response to a particular pathogen.

Using genetics to define differences between species

We get around these problems by looking at genetic differences between species. New sequencing technologies make it relatively quick and easy to sequence the genome of an individual. 

In a typical genome, around 25 000-50 000 genes code for proteins. If we want to know how two species differ we can compare sequences of these protein coding genes and see which types of gene differ the most between species. These changes in gene sequences mean the proteins work differently.

Not all genes are expressed, that is translated into proteins, all the time, and we can see which sets of genes are expressed at particular times and in different organs, like petal or leaf. So when looking at how species differ, we can also look at which genes show the biggest changes in expression level, which would mean one species having more of a particular protein than the other.

Having a list of which genes show sequence changes and which show expression changes is not much help if it’s just a list of genes xzyabc and rst. What really makes this a useful technique are the huge databases which have been built up over the past 20 years. Work in model species such as yeast, Arabidopsis, mouse and Drosophila have determined functions for many genes in typical genomes. We can match the genes in our ‘interesting genes’ lists to sequences from these model organisms to find out that, for example, gene xzy looks like a disease resistance gene, or gene abc looks like a gene that controls root growth rates.

A typical comparative study might highlight hundreds of genes which differ between species, so even with good descriptions of function we still have a lot of data to sift though to find patterns. We can simplify the lists by using GO (Gene Ontology) terms. This is a way of describing what genes do in a very defined way. 

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A tale of two models

James Lloyd (University of Leeds) was the lead author on last month’s Plant Journal paper on nonsense-mediated mRNA decay, which demonstrated that working on a model plant can sometimes be a hinderance rather than a help. In this guest post he explains how he and his supervisor Brendan Davies overturned widely held assumptions about NMD in plants – by working on moss.

Physcomitrella patens, the model moss, growing on agar.

Plant biology has been greatly advanced by the use of Arabidopsis thaliana as a model. Fantastic community resources, such as mutant collections and numerous genome sequences from natural accessions have aided researchers in all areas of plant sciences. However, A. thaliana is far from perfect. Nonsense-mediated mRNA decay (NMD) is a little heard of pathway to regulate transcript stability and it has recently been shown to be involved in pathogen response in A. thaliana (Rayson et al., 2012).

The mechanism of mRNA decay has largely been worked out in animals and revolves around the phosphorylation of an RNA helicase called UPF1 by a kinase SMG1. However, fungi and A. thaliana both lack the SMG1 kinase, so it has been a mystery how the fungal and plant proteins are phosphorylated. We have recently shown that SMG1 is not animal specific but is an ancient component of the NMD machinery, appearing in the genomes of all plants examined, with the exception of A. thalianaSMG1 is even found in the genome of A. lyrata, a close relative of A. thaliana.

Arabidopsis thaliana, the model plant.

The lack of SMG1 in the genome of A. thaliana meant that we had to use another plant as our model to understand the role of SMG1 in the plant kingdom. Enter moss! Physcomitrella patens is the model moss, with a sequenced genome and a high rate of homologous recombination it is a relatively simple task to identify and delete any gene in the genome. The rate of homologous recombination is much lower in flowering plants than it is in moss, meaning that short (around 1 Kb) of moss genomic DNA from upstream and downstream of a gene of interest can be cloned around an antibiotic selection gene and then transformed into moss cells and a large proportion of antibiotic resistant plants have the gene of interest deleted.

We deleted SMG1 from moss and found that NMD was compromised thus placing plant SMG1 in the NMD pathway (Lloyd and Davies, 2013). Therefore, many plants including crops like rice and maize are likely to rely on SMG1 to control gene expression through NMD. Future research will hopefully reveal why A. thaliana has lost SMG1 and if another kinase has replaced SMG1 in this plant.

A. thaliana has been useful in characterising other components of the NMD pathway in plants, such as UPF1 and understanding the biological role in plants (such as controlling the pathogen response). However, it was limited in helping us understand the NMD pathway of commercially important crops and it was moss to the rescue!

Animal researchers have long used multiple, evolutionarily diverse models, including but not limited to fruit flies, C. elegans, zebrafish, frogs and mice. Despite big differences between invertebrates like fruit flies and vertebrates like humans, a great deal about human biology has been learnt by using these organisms in the lab.

Many important questions cannot be answered by simply studying A. thaliana, big differences in fundamental processes exist between accessions. Therefore, multiple models are needed in plant sciences. Moss is just one plant that can help compliment research in other plant species, using the powerful genetic tool of homologous recombination.

References:

Lloyd JPB and Davies B (2013) SMG1 is an ancient nonsense-mediated mRNA decay effector. The Plant Journal 1365-313X http://dx.doi.org/10.1111/tpj.12329

Rayson S, Arciga-Reyes L, Wootton L, De Torres Zabala M, Truman W, et al. (2012) A Role for Nonsense-Mediated mRNA Decay in Plants: Pathogen Responses Are Induced in Arabidopsis thaliana NMD Mutants. PLoS ONE 7(2): e31917. doi:10.1371/journal.pone.0031917

Images courtesy of James Lloyd. 

Staying together: green beginnings

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Published on: November 5, 2013
This seaweed, Ulva linza, would only exist as undifferentiated cells if not for bacterial signals

The second of our series of blog posts Celebrating Basic Plant Science is written by Juliet Coates, Lecturer in Molecular Genetics at the University of Birmingham.

Living organisms can be categorised in a number of ways, but one very obvious “either/or” distinction is between organisms that are made up of a single cell – unicellular organisms – and those that are many-celled, or multicellular.

The multicellular state arose many times during evolution: animals, plants, algae, amoebae, fungi and bacteria can all be multicellular. Multicellular organisms completely underpin life on Earth as we know it today – and they all must have evolved from single-celled ancestors. We understand a little of why they might have done so, as being multicellular gives a number of competitive advantages: increased size and improved nutrient collection being just two. Yet how multicellular organisms came to be is a key biological problem that is still largely unanswered.

I am a plant scientist, so I am particularly interested in the origins of multicellular green things: plants and algae. Without becoming multicellular, plants would never have colonised the land, and the evolution of multicellular plants and algae was key in shaping our climate, our ecosystems and our oxygen-rich atmosphere. How green multicellularity arose seems to me to be a really fundamental thing to understand, but it is a little-addressed question. Here I’ll give an overview of the important findings to date about the evolution of multicellularity.

 

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Minding the scientific skeleton in the closet

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Published on: October 22, 2013

Two weeks ago, Pamela Roland from UC Davis retracted a Science paper originally published in 2009. She gave her perspective on the events leading to the retraction in a Scientific American blog post. Here a post-doc in her lab gives his side of the story, emphasising the importance of teamwork, honesty, and willingness to speak up at every level of the academic hierarchy.

Benjamin Schwessinger obtained his PhD at The Sainsbury Laboratory, under GARNet Advisory Committee member Cyril Zipfel. He joined Pamela Roland’s UC Davis group in 2011. This post is an edited version of DO mind the scientific skeletons in the closet, originally published on the blog he co-hosts with Ksenia Krasileva, a postdoctoral fellow in Jorge Dubcovskey’s lab.

 

Yet another Science paper retracted today. Nothing much new unfortunately – except that for me and my colleagues this was not just another example of non-reproducible work. We planned our research projects around it. So here are my thoughts about this, and just in time for Halloween.

There has been much talk about the problem of reproducibility of data and the rise in retractions. The discourse is mostly centered around the perpetrators and the negative impact this sloppy or knowingly flawed science has on the industry and the perception of scientific endeavor in society: Who did this awful study? What reviewer did not catch this missing control, the sloppy stats? Why don’t they admit their mistake? What’s wrong with peer-review? Well, much is wrong with the industry, and many people have great ideas about how to tackle some of these issues.

What is usually missed from the discussion is the impact such dubious science can have on young, early career scientists. What do you do if you come to a famous (or not so famous) lab, you get a project, which is based on fantastic data and high impact publications, but you cannot reproduce it? How do you approach this issue? What does it mean for you and your career if your project goes to shreds because it’s based on bad data? Do you get another chance? Will you be forever associated with this flawed data and your reputation damaged? Will you get so disillusioned with science to the point that you leave academia? Do you try to fix the problem or silently move on? Do you tell the boss and explain your situation? Or is it you? Can you just not get it to work? Can it really be that this is all wrong? Why cannot I reproduce this data? And the questions go on and on … I think everyone can appreciate the complexity of the issue. Many people I talked to had their own experience to share.

Here is what’s frightening:  sloppy science and misconduct, I thought, is something you read about in journals and not something I would experience myself. This would never affect me or close friends in other labs, who are all great scientists in my eyes. I was wrong. (more…)

Celebrating Basic Plant Science: Siobhan Braybrook

The first in our series of Celebrating Basic Plant Science articles comes from Siobhan Braybrook, a Career Development Fellow at the Sainsbury Laboratory at the University of Cambridge. She explains her work on plant development and discusses why she thinks basic plant science is value for money. 

In parts of India people have built ‘living bridges’ with traditional methods. Could developmental biology build the living bridges of the future?

How do we measure the importance of scientific works? Do we require immediate applications? Do we simply need to know? Both basic and applied science are important and vital for our sociological and scientific progress, but we tend to measure their impact with a very immediate and short ruler, one which is biased towards applied outcomes. Basic science is concerned with knowledge for knowledge’s sake, the desire to know. Applied science is directed towards a specific problem and it’s solution. Here, I propose that is impossible to anticipate the value of a basic scientific work beyond its immediate context, and that attempting to do so might just force us to narrow our field of imagination and innovation.

My group focuses on a basic scientific question- we would like to know how plants grow shapes. Our research definitely falls into the category of basic science as we pursue the answer to this question, not with a specific application in mind, but with a simple desire to know. But that does not mean that we don’t find applied directions during our pursuits.

Plant cells are pretty special to me because they exist in a box; the plant cell wall contains all of the other cell contents, allowing the cell to attain high pressures and also being the regulator of cell shape. We use biology, genetics, biochemistry, and materials science to understand how the cell wall controls cell, organ, and whole plant shape. As an example, we have shown with collaborators in France that new organ formation strictly requires a particular change in the cell wall, altered pectin chemistry. It was surprising that something as simple as pectin, the same thing used to make jellies set, was able to control whole plant shape by limiting new organ growth. These experiments have directed us to look at other growth processes that might be controlled, in part, by pectins in the cell wall.

From a basic science standpoint, our findings were very satisfying- we had found out something new and interesting. But they have also led us down some less familiar paths, into the realm of applied science. Can we take what we have learned about a biological material, the cell wall, and design man-made materials that also have the potential to grow? Could we one day place a small block of material on the ground and have it grow into a house? A car? Alternatively, if we understand how the cell wall controls growth, could we plant a seed that grows into a house frame? A chair? It is unlikely that any company would touch this idea without a very, very, very long pole at this time. It is too speculative, maybe even too crazy. But within the realm of basic science, we can continue to chip away at the possibility- with a freedom that does not require a final product right away, a freedom that allows us to grow our ideas along side our plants.

In closing, it is probably highly simplistic to separate basic and applied science. There is cross talk between the two, research projects that exist in a continuum, and research questions that are entangled. However, there are some very special things about basic science: you don’t need to know exactly where you are going in order to end up somewhere cool; you can explore things for the sake of knowledge which gives a lot of freedom; and sometimes you find out unexpected things that end up having massive applied impacts that you might never have anticipated. It is essential that we create a place for such scientific freedoms, that we don’t assume which pursuits have value before they have been investigated, and that we allow for the possibility of novel discoveries.

You can read Siobhan Braybrook’s research about pectin and new organ formation in Braybrook and Peaucelle 2013, PLoS ONE 8(3): e57813 and Peaucelle et al. 2001, Curr. Biol. 21:1720

Image credit: Screwtape via Flickr

 

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