The UEA Small RNA Workbench: New and improved tools for high throughput small RNA analysis

RNA silencing is a complex and highly conserved regulatory mechanism that is involved in diverse processes such as development, pathogen control, genome maintenance and response to environmental change. Since its recent discovery, RNA silencing has become a fast moving area of research of great importance in both plant and animal molecular biology. Its discovery has not only been a major breakthrough for our understanding of complex regulatory processes in living cells, but has also opened the door to the development of novel therapeutics for human diseases. The award of the Nobel Prize for medicine highlighted the importance of these findings in 2006 for the discovery of RNA interference. Small RNA molecules (sRNAs) play a crucial role in the RNA silencing machinery in that they can interact with other nucleic acids in a sequence specific manner to mark them as targets for inactivation.

To learn more about this mechanism and its implications, researchers employ cutting-edge sequencing technologies to obtain huge datasets of sRNA sequences (up to 200 million sequences about 20-30 nucleotides in length) from a number of organisms. With these technologies becoming more accessible and affordable, it is now possible to obtain multiple large-scale data sets, which allow the analysis of changes in complete sRNA populations. These changes can indicate the involvement of RNA silencing pathways in biological process of interest, such as regulation of key developmental stages in an organism.

In previous BBSRC-funded work, we have addressed some of the challenges involved in analysing massive sRNA data sets through the development of the UEA sRNA workbench. This is a downloadable program that has attracted both academic and commercial users, and that allows biologists to identify key sRNAs in their data sets without bioinformatics support. Since its introduction in 2012, the workbench has been downloaded over 4,000 times and the website receives around 50 requests for the RSS feed per day as its user base rapidly expands. However, there are several new features and improvements that the workbench requires whose need are being driven by new research in sRNAs, user feed-back, and the rapid development of ever deeper and cheaper sequencing technologies. In this project, we will build on our publicly available software tools for sRNA analysis, with the aim of providing new cutting edge tools, both locally and through the cloud-computing, so as to continue to provide a world-leading solution for the analysis of plant and animal sRNA datasets. This will include the addition of new tools for preparing and accessing the quality of sRNA sequence data, predicting new classes of sRNAs, and for analysing sRNA data in tandem with RNA-seq data. This will provide biologists with much needed resources to help identify novel sRNAs and their function in order to direct further wet-lab studies.

High throughput sequencing technologies such as Roche 454 sequencing, Illumina sequencing, and ABI Solid have become an important tool for researchers in the field of RNA silencing, since they can sequence tens of millions of small (s)RNAs in a single experiment. This makes it possible to produce snapshots of the complex sRNA populations of living cells and hence gain insights into the production and function of this important class of regulatory non-coding RNA molecules. With sequencing technologies becoming ever more affordable, it is now possible to follow changes in sRNA signatures through time series or to compare the effects of different treatments or different tissues at a high resolution.

In previous BBSRC-funded work, we have addressed some of the challenges involved in analysing massive sRNA data sets through the development of the UEA sRNA workbench. This downloadable program has attracted both academic and commercial users, and allows biologists to identify key sRNAs in their data sets without bioinformatics support. However, there are several new features and improvements that the workbench requires whose need are being driven by new research in sRNAs, user feed-back, and the rapid development of ever deeper and cheaper sequencing technologies.

In this project, we will build on the sRNA workbench, with the aim of providing new easy-to-use, cutting edge tools, both locally and through the cloud-computing, so as to continue to provide a world-leading solution for the analysis of plant and animal sRNA datasets. Moreover, we will continue to make the workbench available for download as command-line based tools so that expert bioinformatics users can easily integrate them into pipelines. The workbench will incorporate new algorithms that will be designed to perform analyses of multiple-sample sRNA datasets and also relate them to RNA-seq data. It will also include a new continuous data stream system suited to memory intensive tools in the workbench.

Since its release in early 2012 the sRNA workbench has been used extensively by researchers from around the world with over 5,900 unique visitors and more than 4,000 downloads. It has an attractive and easy to use interface and supporting website and, to our best knowledge, the workbench is the only freely available complete downloadable solution for processing and analysing high throughput sRNA data. To cater for our user base and maximise impact on the sRNA community, we use a variety of social media, for example, twitter, RSS delivery of direct comments on the website, and emails. Through these methods we regularly receive requests for new features and tools such as those that we propose to implement in this project, and publications are appearing in which the tools are cited and used as a major component of the analysis. It is therefore clear that user demand for tools such as those to be developed in this project is strong and growing.

Throughout the development of the sRNA workbench, Moulton's group has consistently released new tools and updated them often and early to ensure that the wider public has access to our latest work as soon as it is available. Continuing with this theme, all algorithms and software generated in this project will be made freely available and released in a similar way.

As part of the project, we will also make the workbench available through cloud-based systems, such as Galaxy and Illumina's Basespace genomics cloud-computing resource. This will ensure that both academic and commercial users will be able to take advantage of the tools we create. Indeed this open philosophy has already generated interest from various commercial organisations which we will continue to pursue. These include recognition from the UK based bioinformatics consultancy firm Eagle Genomics in their "Elements of Bioinformatics", as well commercial tool usage in various institutions worldwide such as the Beijing Genomics Institute (China), Bayer CropScience (France) and Xcelris Genomics (India).

Members of Moulton's group have previously promoted the sRNA workbench at several conferences and commercial events, including: Plant and Animal Genome Conference (PAG), USA, The RNA Workshop, Spain, UK Next Gen Sequencing, Nottingham University, various talks given to the SIROCCO European consortium, Genomics Automation Europe, and The Keystone Symposia. We will continue with our program of advertising through these avenues as we feel this is an important way to promote our resource to the research and commercial communities.

The sRNA workbench has already been utilised as a resource for various teaching activities, including a series of four UK-based practical courses. In addition, the website contains several downloadable video tutorials for using the workbench tools. Our proposed collaborations with The Genome Analysis Centre will also open up new possibilities for cloud- based training activities. In these ways, we will continue to promote use of the sRNA workbench by the widest possible audience and also address the need for tools and training for the next generation of biological researchers.

As the sRNA workbench has been specifically designed to make it easy to maintain and extend, it will be possible for future development of new features to be driven by short-term projects. In particular, PhD projects could lead to the development of new tools required by the community. Such projects will facilitate training of PhD students in the process of developing and promoting cutting-edge research to a broad user base in the increasingly important area of bioinformatics.

We expect that the proposed tools and improvements will ultimately contribute towards improving quality of life through enabling molecular biologists to understand and exploit key molecular pathways in important crop plants and animal models (e.g. tomato and chicken), as well as to develop new RNA based therapeutics for diseases such as cancer.