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

Comments: No Comments
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…)

In pictures: Software Carpentry for Plant Scientists

Comments: No Comments
Published on: April 11, 2014

Over 30 plant scientists gathered at the University of Warwick this week for our Software Carpentry for Plant Scientists bootcamp. Together we learned to move through space and time using Git, how to make, explore and delete files and directories, how to use Version Control and how to program defensively. As ever we encouraged everyone to Tweet about the event and we’ve collected the tweets in a Storify, which you can access here: sfy.co/ffpW

Some photos from the event are below – enjoy! The photo on the second row is the team that made the event such a success. From left to right: Jason Piper, Charis Cook, Leonor Garcia Gutierrez, Aleksandra Pawlik, Christina Koch and Lisa Martin. Thanks especially to our trainers Aleksandra (sent to us from the Software Sustainability Institute, UK) and Christina, who came all the way from Vancouver.

GARNETSWC

PlantSci 2014 and Plant Science Careers

Categories: resource, UKPSF
Comments: No Comments
Published on: April 8, 2014

The GARNet team travelled up to York last week for the PlantSci 2014 conference. It was a fantastic event and I highly recommend it for future years. The variation between talks, which were all perfectly pitched for a general plant science audience, made the sessions exciting and maintained everyone’s interest.

A highlight of the conference was the Panel Discussion on the Future of UK Plant Science. The Panelists – Mike Bushell, Mark Chase, Sarah Gurr, Sandy Knapp and Dale Sanders – responded to the Status Report (download the PDF here) and spoke briefly about what they felt were the most important challenges for the UK plant science community to deal with.

To me, the most significant issues were put forward by early career researchers from the floor. The Panel and the report, which drew data from a community-wide survey, emphasised skills shortages and a lack of young talent entering the field; but several young researchers present spoke out about lack of support for those young scientists that are working in the field.

One person on a PhD program with funding for a short internship in industry or policy found it difficult to find a placement related to plant science. Two final year PhD students from very different research backgrounds spoke of their frustration in not knowing where to look for post-doctoral jobs. Despite being highly trained in areas within the ‘skills gap’ often referred to in reports, including the UKPSF report, they felt that academic post-doc positions (and the uncertain future that comes with them) were the only options they had.

If you feel passionately about education, training and/or plant science careers and career paths, see UKPSF working group call document (PDF) on information on what the UKPSF is doing to tackle these challenges. There will be UKPSF working groups on Training & Skills, Funding, Portfolio Balance, Regulation, and Translation.

Looking for a plant science job? (more…)

Report launch: Developing Plant Synthetic Biology in the UK

Categories: GARNet, synthetic biology
Comments: No Comments
Published on: March 31, 2014

plant synthetic biology

 

We’ve been working on the meeting report from last year’s An Introduction to Plant Synthetic Biology workshop for months, so we’re delighted that GARNet Chair Professor Jim Murray is going to launch it tomorrow during his talk at PlantSci 2014!

You’ll be able to download the report, Developing Plant Synthetic Biology in the UK: Opportunities and Recommendations tomorrow on the GARNet website, or if you’re at the conference come and see Jim, Lisa or Charis to get a printed version.

To keep up with all the news from PlantSci 2014, follow #PlantSci2014

Plant Doctors at the Big Bang Fair

Comments: No Comments
Published on: March 19, 2014

BigBang cover2

On Friday I went to the NEC in Birmingham to join an intrepid team of plant scientists running activities on an exhibit at the free national science, technology and engineering event the Big Bang Fair. The stand was organised and funded by the British Society of Plant Pathology and the John Innes Centre.

The theme of the exhibit was Plant Doctors. Visitors learned about plant diseases and were encouraged to think about a world without plant doctors – what effects would uncontrolled plant diseases have on our air quality and food supply?

There were four activities on the stand, aimed at different age groups.

The main event was the Plant Doctors activity, where children and young people could don a white coat and clipboard and learn to diagnose a bacterial, fungal or viral plant disease. In my experience the particularly gruesome crown gall tumour (just like the black death!) went down well, but some groups were fascinated by the spores on the bean rust pustules (little mushrooms), which we showed them down the microscope.

At the Polling Station, adults and young people discussed the benefits and drawbacks of bio-control, pesticides and GM approaches to controlling plant pathogens. Older Plant Doctors were also invited to vote on how best to treat the plant diseases. I didn’t spend much time on this activity, but it seemed to keep some groups very engaged. It was certainly an effective way of talking about the importance of innovation in plant science. (more…)

Recently in the GARNet Community … (4)

Categories: Arabidopsis, GARNet
Comments: No Comments
Published on: March 11, 2014

Software Carpentry countdown

It’s been all systems go for our Software Carpentry bootcamp, which is getting startlingly close now – just over a month away! We’re delighted to have confirmed another sponsor, the Company of Biologists, with thanks to Journals Development, Journal of Cell Science, the Journal of Experimental Biology, Disease Models & Mechanisms and Biology Open.

We’re also pleased to confirm our instructors, Aleksandra Pawlik and Christina Koch, who are fine-tuning the programme at the moment. We’re very grateful to our local helpers too: Leonor Garcia-Gutierrez, Krzysztof Polanski and Jason Piper, all PhD students from the University of Warwick.

 

Congratulations

Two plant scientists from our community became Royal Society of Edinburgh Fellows last week. GARNet Advisory Committee member David Salt (Aberdeen) and former Committee member Claire Halpin (Dundee) are two of 53 new elected Fellows. David Rae of the Royal Botanic Garden Edinburgh was the only other plant scientist on the list. Congratulations to all of them!

 

Jobs and workshops for early career researchers

This must be prime plant science recruitment season, because there are a lot of post-doc and other job opportunities out there at the moment – Oxford Brookes, Warwick, Birmingham, John Innes Centre and East Malling (not Arabidopsis) research are all recruiting. Further afield, I’ve spotted Arabidopsis research post-doc vacancies in Wageningen and Alabama.

 

Synthetic Biology review

Chloe Singleton presented her research on synthetic metabolons at our Plant Synthetic Biology workshop last year – you can see slides from her presentation here. A review article on the subject from Singleton and her colleagues is out now under Advance Access at the Journal of Experimental Biology (doi: 10.1093/jxb/eru050). The review discusses types of synthetic metabolons that have been built in vivo and in vitro to date, and explains how they might be used in chloroplasts to improve photosynthetic efficiency.

Jorge Cham and the Power of Procrastination

Categories: something fun
Tags:
Comments: No Comments
Published on: March 6, 2014
 "Piled Higher and Deeper" by Jorge Cham. www.phdcomics.com
“Piled Higher and Deeper” by Jorge Cham. www.phdcomics.com

Jorge Cham, creator of PhD Comics and legend among post-grads, spoke at the University of Warwick last night as part of his UK tour.  His lecture was essentially a stand-up gig, complete with theatrical pauses and audience interaction, but it did have a serious point: stress and guilt are bad for your health and for your work, so embrace the power of procrastination!

Over this morning’s procrastination, I came across this Guardian article on mental health among PhD students. It has a message surprisingly similar to Jorge Cham’s ‘comedy’ lecture. It argues that mental health problems, from stress to suicidal thoughts, are an accepted part of the PhD process.

Though Cham did not directly refer to serious mental health problems in his talk, he constantly talked about the stress and worries common to PhD students: my supervisor thinks I’m an idiot, why can’t I get anything done, when will I finish. To make it worse, these are often accompanied by lack of money, little sleep and bad food.

In typical academic fashion, Cham did not offer empty words of wisdom or advise talking about your feelings. He presented facts on the Power of Procrastination (the title of his talk).

There is scientific research suggesting procrastination can be a positive thing. I didn’t note the reference Cham used, but Chu and Choi (2005; J. Soc. Psychol. 145:245) is an accessible review. ‘Active’ procrastination as opposed to ‘passive’ procrastination, which Cham labeled laziness, can be an effective way of dealing with a problem. Interestingly, for some people procrastination is a good way of coping with stress!

Then there are the numerous examples of highly successful procrastinators. I don’t want to ruin Cham’s punchlines, so I’ve come up with two well-known biologist procrastinators. Francis Crick took 15 years between undergraduate and post-graduate qualifications – although during that time the Second World War happened – and Alexander Fleming famously discovered penicillin by accident. I like to think of him stumbling upon that fateful petri dish while writing a detailed 12-step plan to tidy his messy bench instead of doing real work.

Cham’s comics normalise the everyday struggles every PhD student faces during a time they hoped would be enjoyable, stimulating and life-affirming. Some of the most common comments Cham receives are thanks for making post-grads feel they are not alone in feeling stupid, stressed and fearful of the future, failure, and their supervisor. The comics, like his talk, encourage students not to be overwhelmed any of that – nor to feel guilty about using the power of procrastination. Or going to seminars largely for the free food.

Opportunities in plant science, via social media

Categories: Arabidopsis, Workshops
Comments: No Comments
Published on: March 5, 2014

I don’t usually put this kind of thing on the blog – it’s prime mailing list fodder. But I was writing a round-up post and this section got more than long enough for its own post!

If you’re on our ArabUK mailing list, you’ve probably noticed a flurry of activity this week. There are a lot of post-doc and other job opportunities out there at the moment – Oxford Brookes, Warwick, Birmingham and East Malling research are all recruiting. Further afield, I’ve spotted Arabidopsis post-doc vacancies in Wageningen and Alabama.

There are also two  fully funded workshops open to applications from early career researchers at the moment. Erik Murchie is co-organising a workshop in Thailand (Thailand!) and is looking for post-docs who work on abiotic stress physiology and genetics. Successful applicants will work with rice researchers from Thailand at a 4-day workshop, with the aim of improving understanding of oxidative stress in rice. Apply by 1 May.

Secondly, this synthetic biology summer school in Berlin looks like a great opportunity for PhD students and post-docs working on, or are on the periphery of, synthetic biology. It sounds like an interesting 5 days discussing how to ‘evaluate new techno-scientific areas’ and ‘analyse the societal dimensions of synthetic biology’. As an aside, if you get selected I highly recommend the Alternative Tour of Berlin – it might even give you some ideas for the workshop! This one is a tight deadline, applications close on 10 March.

All of these, including the job at Warwick, for which the advertiser is across the corridor from me, crossed my path on Twitter. If you’re looking for a job or training opportunities and/or want to keep up with news from the community, Twitter is definitely a good place to start. Just follow the right people – try the GARNet accounts (obviously! Me, Lisa, Ruth) and also Mary Williams, Anne Osterrieder, BSPP and UKPSF. Anne even has lists of categorised tweets, a great place to find relevant Twitter users.

You can just use Twitter for harvesting information. You don’t even have to fill out your profile, though you do need a username. But it can be a valuable tool for networking and the ‘branding’ that careers advisors sometimes talk about. Anne has a great paper in Plant Methods about how to use social media as a plant scientist. I use it to share plant science and occasional sci-fi links I come across that I think others will find interesting – if I read an article or paper, attend a good talk, or see a plant science job opportunity or conference.

«page 1 of 22
Welcome , today is Monday, April 21, 2014