Genome Resequencing for Mutant Identification

As most biologists will be aware, the cost of DNA sequencing has been falling well in advance of the costs predicted by Moores law (although argued by Neil Hall a few years ago, this might not have been the best thing to happen, intellectually at least).

Instead of simply sequencing many genomes for the sake of it, this also offers opportunities for researchers to use this technology to ‘do-science’ that might previously have been prohibitively laborious or expensive. One such area where this is true is in the identification of novel mutations in plants, especially in Arabidopsis.

Classic approaches to identity the location of an EMS mutation involved mutant identification, backcrossing, selection, rough mapping by PCR or CAPS markers, probably more crossing and then a little guesswork toward the end..…..before using Sanger sequencing to identify what you hope is the causative mutation. Even with a strong following wind this process could take upwards of a year……. many a 1990s PhD thesis was written off the back of mutant identification. In contrast it is now relatively cheap to resequence the Arabidopsis genome so a lot of time can be taken out of this process. In addition, resequencing can remove some of the difficulty involved with selective of mutants that have a subtle phenotypes wherein inaccurate selection of putative mutants would significantly set back the process.

Back in 20111, Anthony Hall’s group in Liverpool University used resequencing in parallel with classic genetics to identify the lesion in the novel early bird1 gene (ebi1), which has a defect in function of the circadian clock. In this case ebi1, which was generated using EMS, was backcrossed 4 times to reduce the number of EMS-induced SNPs not associated with phenotype, and then sequenced alongside the original wildtype plant (from the WS ecotype). The critical part of the protocol came in the power of the software they used to detect homozygous SNPs in the ebi1 line. Indeed the researchers ran into some difficulties due to a high number of SNPs they initially identified. However, when they combined altering the stringency of SNP-calling together with classical rough mapping they were left with approximately 30 SNPs to finally assess. Using a priori knowledge of proposed gene function and by investigating expression changes in these candidates they ultimately identified a novel mutant. Although this process was ultimately successful, it took some extra time due to the difficulty of mutant selection, optimization of the SNP-calling software and subsequent analysis of gene expression.

A recent paper from the lab of Lucia Strader at Washington University in St Louis shows how powerful resequencing can be if you are using a robust method of mutant selection. In their case they isolated mutants with a defect in the root growth response to ABA, which is an unequivocal phenotype to score. They backcrossed their initial mutants, selected for ABA resistance in F2 generation before resequencing these resistant plants. Using this process the authors report that they narrowed their search to between 3-10 candidate genes and that they have subsequently identified novel (unpublished) genes using this method. In addition, as an exemplar of their protocol they used it to isolate novel alleles of known ABA-resistant mutants.

Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167
Schematic for mutant identification using NGS. Reproduced from Taylor and Francis PSB http://dx.doi.org/10.1080/15592324.2014.1000167

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

In parallel they used a similar protocol to the Hall lab where they resequenced non-backcrossed plants and then selected SNPs that only lay within exons.Using this approach they identified between 100-200 homozygous SNPs, a potentially fifty-fold increase compared to their other method. Therefore when you are working with a strong robust phenotype it is probably worth the extra time to obtain a back-crossed population in order to have greater confidence you are isolated your mutant of interest.

The authors importantly note that one limitation of this protocol is that by only selecting for exonic mutations, they are removing the possibility of identifying mutants with splicing or non-coding defects, which may in turn rule out a number of candidate genes.

 

For me the take-home message from this second study is that if you have a robust phenotype to select for and are confident that your mutation is novel then use of ever-improving NGS is now a time and cost effective way of mutant identification.

In fact this technology might inspire a return to the forward genetic screens of the 80s and 90s , with the aim of identifying novel genes involved in well characterised signaling pathways……..except that PhD students might now have to characterise 10 novel genes prior to graduation….

Multi-scale Biology Meeting.

What is Multi-scale Biology?

That was the overriding question that occupied attendees of the inaugural meeting of the Multi-scale Biology Network, held at the University of Nottingham on June 1st 2015. This newly funded BBSRC network brought together biologists, physicists, engineers, and the funding bodies (amongst others) for this meeting that aimed to set the agenda for this area of future collaboration. Storify of the meeting.

Those who had also been involved in the genesis of synthetic biology policy might have felt a touch of deja-vu over the questions being asked at this meeting. However whereas the term ‘synthetic biology’ might have remained opaque after those early meetings, the feeling at the end of this day was that good progress had been made into an understanding of what this network might achieve.

Although there were <a href="http://www.multiscalebiology.org.uk/events/event/springboard/" onclick="_gaq.push(['_trackEvent', 'outbound-article', 'http://www cialis 20mg en france.multiscalebiology.org.uk/events/event/springboard/’, ‘speakers from a range of biological disciplines’]);” target=”_blank”>speakers from a range of biological disciplines there was no requirement for forced interactions between different research areas. Instead there was a sense of ‘wondering what can be learnt‘ when for example, plant scientists talked to neurobiologists…

The meeting was kicked off by Professor Markus Owen from the Nottingham Maths department and the agenda had been clearly designed to encourage significant amounts of discussion, be it during the lecture period or over an extended lunch in which attendees split into small groups to discuss challenges in MSB.

Many of the attendees were involved in some form of systems biology so the sense was that MSB comes as the natural extension of that area of research. Instead of looking at a single network, how should researchers extend their thinking to include how these networks interact at different scales…..without losing the quality of analytic data?

Professor Alfonso Martinez-Arias (Cambridge) led the first discussion and made the important point that even though we now have enormous amounts of data, what is it we actually want to learn?….and is focusing on that type of question compatible with current necessity for publication in high quality journals?

Immediately after lunch Professor Carole Goble introduced a new ERASysApp network (FAIRdom) in which she is involved that aims to ensure that systems biology projects make their data, operating procedures and models, Findable, Accessible, Interoperable and Reusable (FAIR). This was a timely reminder that it is very important to produce data that is reusable by the community so that people aren’t reinventing the wheel in each experiment. In 2016 GARNet will be hosting a meeting with the Exeter Centre for the Study of Life Sciences that will address issues surrounding data reuse. Details to follow later in 2015!

Toward the end of the day Professor Martin Howard from the John Innes Centre led the final discussion that attempted to coalesce the thoughts of attendees. One suggestion was for biologists to act ‘like engineers’ and to use ‘dirty tricks’ as to get tasks completed. Understanding the details can come later…. getting the job done is most important….. whatever the job is that needs to be done! It was also discussed that coming to any ‘effective theory’ of biology is almost impossible given the unpredictable nature of the field.

The results of group discussions!

The results of group discussions!

Overall it seems that making any decisions about how multi-scale biology projects can be implemented will depend on the funding environment. To that end Michael Ward from the EPSRC stressed that mathematical biology is important for mathematical science funding and urged attendees not to be put off from looking in their direction. Ceri Lyn-Adams from the BBSRC informed the group that although there wasn’t any money specifically ring-fenced for MSB, they welcome applications in this area under existing mechanisms.  What was clear is that MSB and systems biology remain in the forefront of BBSRC funding strategy!

By it’s very nature MSB will require large projects that might bring together a few responsive mode-size projects under a single umbrella. Hopefully new researchers will be able to be involved in this type of project and the money doesn’t all go to large established groups.

Plant Scientists were well represented at the meeting most notably by Martin Howard, Malcolm Bennett and Leah Band, who gave the final talk of the day. The multi-scale nature of plant biology was highlighted in this more than any other talk, as Leah discussed biology at the organismal, tissue, network, cellular and enzyme level. Her work builds the prior knowledge of GA transport and biosynthesis to make mathematical models to predict tissue expansion.
Multi-scale Plant Biology

Importantly for the GARNet community, the take home message from the meeting was that plant scientists will play a major role in the future of multi-scale biology so once the funding opportunities are revealed they should not be reticent in submitting bids.

Arabidopsis Information Portal at PAGXXIII

Last Monday the Arabidopsis community gathered for the Arabidopsis Information Portal workshop at PAG XXIII. The Arabidopsis Informatics Portal (AIP) was funded by NSF and BBSRC to move beyond the Arabidopsis genome resource provided by TAIR toward linking the genome to the epigenome, proteome, transcriptome and interactome.

AraportThe first talk was a short update from Eva Huala, formerly of TAIR and now of Phoenix Bioinformatics, the nonprofit company she started in order to keep TAIR going. Huala explained that after TAIR’s NSF funding ended, the pay-to-access model was chosen over the alternative pay-to-submit (open access) approach. This means TAIR is focussed on ensuring the subscribers get the best possible value for money by providing the best possible database curation, manual annotation and user experience. Most TAIR subscription fees are paid by libraries, as if it was a journal, but researchers from institutions whose libraries do not pay the fee will be able to access TAIR’s manual annotation after a year’s embargo.

Next, Sean May (NASC, University of Nottingham) explained that NASC is a module of AIP and is currently integrating with the ABRC. He is consulting the community about the development of NASC, so make sure you have your say in the NASC Strategy Survey: http://bit.ly/1J24Hgk

Chia-yi Cheng (JCVI) gave an overview of Araport, the online home of the AIP. Araport federates diverse datasets from other places, for example TAIR, UniProt and BAR, and maintains the Col-0 ‘gold standard’ annotation. It uses JBrowse as the default genome browser and hosts datasets including the CoGe epigenomics resource, which I blogged about last week. (more…)

GEO for plant scientists: Sharing data

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Published on: February 13, 2014

There is currently no microarray service provider in the UK that uploads your plant science microarray data to GEO on your behalf, but publication requires your data to be shared. The most common request from journals is that it is shared on GEO.

GEO has this information page about data submission. While the high-throughput sequence submission guidelines are a still little complicated, microarray experiments have well-established (and enforced!) minimum information requirements and the four main microarray chip providers have customized information pages. An email address is provided for users to email enquiries and ask for help from GEO’s curators.

The Affymetrix page is probably the most useful for UK plant sciences. Spreadsheet-based submission is recommended for Affymetrix deposits, so users should submit an Excel metadata worksheet, CEL files, and processed data for example a Tiling Array. The page gives advice on how to find certain information is given on finding GEO-specific information, and there are template and example spreadsheets.

Once submitted, your dataset becomes a GEO accession and can be identified with a unique accession number. The accession number should be used when you or anyone else references or links to your dataset, which seems like an easy means of tracking its usage within the community.

GEO for plant scientists: How to find Arabidopsis microarray data

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Published on: February 13, 2014

Submission of gene expression data to the Gene Expression Omnibus is now a requirement of publication in most journals, so it is an extremely valuable resource. It is also extremely big, and full of data that isn’t relevant to your question or task at hand – but it is easy to find the right data using the search bar if you follow a few rules. There are example searches on the GEO homepage.

To find data relating to Arabidopsis thaliana, search: (Arabidopsis thaliana[organism])

To find Arabidopsis microarray data, search: (Arabidopsis thaliana[organism]) AND “expression profiling by array”

The easiest way to find other Arabidopsis datasets is to search: (Arabidopsis thaliana[organism]). On the left hand side of the window, there is a ‘Study type’ section. If you click on ‘More…’ a list of study types pops up from which you can select the data type you are looking for (see screen shot below).

You can add any search term you like to the search bar. For example, you could specify author, publication time, types of tissue or stress… or any combination of these. Just keep adding AND in between each term. For example: (Arabidopsis thaliana[organism]) AND “expression profiling by array” AND leaf

GEO provides an informative guide to how to download original records or curated datasets individually or in bulk. You can download data directly from Accession Viewer pages (eg this one) in SOFT, MINiML or TXT formats. Raw data is also available in TAR. You can also do bulk downloads via GEO’s FTP site. All files are compressed using gzip.

It’s also possible to access GEO programmatically in order to, for example, quickly retrieve CEL files from Arabidopsis stress experiments. Again, GEO provide a guide to this, although this is probably something better tackled with some pre-existing knowledge of programming.

GEO post

Register now for GARNet’s 2014 events

We have been busy arranging two great events for 2014! Registration for both Software Carpentry for Plant Scientists (9-10 April) and Arabidopsis: The Ongoing Green Revolution (9-10 September) is now open.

 

On 9-10 April we are hosting a Software Carpentry bootcamp for plant scientists – an Introduction to Programming for Biologists. For those of you who don’t know about Software Carpentry, it is a foundation that teaches good practice in scientific computing, with the aim of providing all scientists with basic, but reliable and transferable, programming skills. If you’ve ever run through the rain to Computing to have a large ChIP-chip dataset split so you can attempt an Excel analysis on it, you’ll know how valuable that is (based on real events – feel free to insert your own experiences there …)!

We’ve worked with the Software Sustainability Institute to develop a programme suitable for both complete beginners and scientists how know their way around the Terminal/Command Prompt but want to improve their skills and learn how to write reliable, re-usable code they can share with their colleagues and collaborators. Registration is £50 and discounted on-campus accommodation is available.

 

Later in the year, the GARNet general meeting is returning for one time only on 9-10 September at the University of Bristol. Our theme is ‘Arabidopsis: The Ongoing Green Revolution’. We have a line up of excellent speakers, including plenary talks from Alistair Hetherington (University of Bristol), Andrew Millar (University of Edinburgh), Rob Martienssen from Cold Spring Harbor Laboratories, and Paul Schulze-Lefert and Maarten Koornneef from the Max Planck Institute for Plant Breeding Research. 

The full line-up and registration details can be found by visiting www.garnet2014.org. More information will appear on there closer to the time of the conference. Registration costs £150 for two days, lunch and refreshments on both days, and a drinks reception on the afternoon of 9 September. We’d also love to see you at our conference dinner on the evening of the 9 September at the Bristol Marriott Royal Hotel (£44 per head for three courses and wine on the tables).

Two GARNet Events

Image by Centimedia.org for GARNet

We have some GARNet news to share!

First of all, we are pleased to finally open registration for the hands-on iPlant training workshop ‘Data Mining with iPlant‘. Unfortunately we’ve had to change the planned location, and it will now be at the University of Warwick. The date is still 17-20 September 2013.

For those who don’t know, iPlant is an incredible free resource which allows its users to access high performance computing power, large scale data storage, and analytical software needed for a variety of data- or compute- intensive research applications.

You can either come for just one day for a free hands-on introduction to iPlant, or stay for four days and get in depth training on how to analyse real data in iPlant. For more information go to: http://www.garnetcommunity.org.uk/news/13-06-19/data-mining-iplant-17-20-september-2013

Our second announcement is more of a save-the-date than an invitation. The GARNet general conference will return next year, possibly for one time only. GARNet 2014: The Past, Present and Future of the Genetic Model Revolution will be held at the University of Bristol on 9-10 September 2014. It will be a celebration of exciting new plant science, and a look at the evolving nature of model systems as well as the brilliant achievements made with them in the past.

The Journal of Experimental Botany kindly recorded and uploaded talks from the last GARNet conference in 2011. Here is Katherine Denby of the University of Warwick talking about the PRESTA project, which since this talk has produced two Plant Cell papers (1,2). You can see the rest of the talks from GARNet 2011 on the JXB website.

Plant synthetic biology round-up

Well, I’ve just about recovered from this week’s GARNet meeting, An Introduction to Opportunities in Plant Synthetic Biology. It was a great two days. For a report of the meeting through the medium of Twitter, including links to resources and papers from the speakers, see this Storify I made – thanks to everyone who Tweeted throughout the meeting!

I’ve rounded up a few of the resources and papers I think would be most helpful for plant scientists below. The Storify of the meeting contains more, and keep an eye on the Journal of Experimental Botany for a series of perspectives and a meeting report over the coming months.

Tools and resources:

  • CellModeller is an open source software from Jim Haseloff’s lab, which allows users to model multicellular systems. It has been used to model the growth and behavious of synthetic microbial biofilms (Rudge et al. 2012, ACS SynBio 1:345), and plant cell division and expansion (Dupuy et al. 2010, PNAS 107:2711). For toll-free links to both papers, go to the CellModeller website.
  • TAL effectors were mentioned in a number of talks, and were presented to the audience by Sebastian Schornack, who declared them fool-proof means of DNA editing. For protocols, papers, and more information see the TAL effectors website, and you can order custom TALs from Life Technologies. Sebastian is keeping a database of papers using TAL effectors on Scoop.it.
  • Golden Gate cloning and its variants are extremely powerful tools for DNA assembly and combinatorial library construction. Speakers Giles Oldroyd and Tom Ellis have used it to great effect. Sylvestre Marrillionet explained to delegates how Golden Gate cloning was invented and what it can be used for – to find out how to use it, see his papers or get in touch with him. This website also gives a good overview and selection of useful papers.
  • Gibson Assembly is another powerful DNA assembly tool, which was presented by Jim Ajioka at the meeting. There is a very comprehensive guide to using it, including sequences and protocols, online here.
  • The Infobiotics Workbench was designed by speaker Natalio Krasnagor. It is a freely available framework for carrying out in silico experiments, from design to results visualisation.

Inspirational plant synthetic biology projects

  • June Medford presented the most complete plant synthetic biology project, the plants which de-colour in the presence of toxins – the synthetic signal transduction pathway that the ‘plant sentinels’ contain is published in PLOS ONE. You can see her papers, many with toll-free links, on her website. Also, if you’re looking for an adventurous post-doc position, she’s recruiting!
  • Last year Giles Oldroyd received funding from the Bill and Melinda Gates Foundation to build synthetic signalling pathways into wheat to enable sybmiosus between this global food crop and nitrogen fixing bacteria. You can see his progress so far in papers on his website.

More information and sites of interest

  • To keep up to date with synthetic biology news and funding, and to link up with possible collaborators, join the Synthetic Biology Special Interest group from the Bisosciences Knowledge Transfer Network.
  • Many of the speakers at the meeting were also at last year’s New Phytologist workshop on synthetic biology. You can see videos of the talks on YouTube, and the meeting report in New Phytologist 3:617.
  • If you’re interested in synthetic biology and want to get plugged into the community, think about going to the 2nd International Synthetic Yeast Genome Consortium Meeting. True, it’s not about green leafy things, but the techniques discussed will be relevant and you’ll make good connections.

Review papers

  • Speaker Tom Ellis recommended this recent review article from Kahl and Endy (Open Access; JBE 7:13) for an overview of available DNA assembly methods.
  • This open access 2012 review by Richard Kitney is an overview of the current situation in synthetic biology – Kitney and Freemont 2012; FEBS Letters 587:2029).
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