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….

Plant Methods reviews NGS for Plant Science

Categories: GARNet, Workshops
Comments: No Comments
Published on: September 12, 2013

Were you at our Tools and Technologies to Advance Plant Research event at Liverpool last year? Well if you were, you can now hear more from three of the speakers in a mini-series of reviews in Plant Methods. And if you weren’t and you’re wondering if next generation sequencing can be applied to your plant research, here’s your chance to find out.

First, Tom Hardcastle wrote a review on using NGS sequence the methylome – it was published in June this year (Plant Methods 9:16). He gives an introduction to the role of cytosine methylation in the regulation of gene expression, and then takes readers through the stages of mapping genome methylation.

Arther Korte’s review on genome wide association studies (GWAS) was published in July (Plant Methods 9:29). He and Ashley Farlow, who are both working on the 1001 Genomes Project, discuss the advantages and disadvantages of GWAS and how to build a good project.

Most recently, a review on NGS for complex crop genomes by Klaus Mayer and colleagues was published (Plant Methods 9:35). They overview various approaches to managing extremely large, complex genomes like the recently sequenced wheat genome. They focus specifically on the Genome Zipper, which Mayer presented at the workshop.

Other workshop talks covered genome-wide association studies, mutant identification, genome mapping, transcriptomics using both ‘traditional’ RNA-seq and new method of direct RNA sequencing, and chromatin mapping. If you want to find out more about using NGS for these applications (it’s not just about nucleotide sequencing any more!), a good place to start is the Tools and Technologies to Advance Plant Research abstract book and the Storify of the workshop, which contains links to a lot more papers and reviews. I also wrote a report for the workshop sponsor, the Genetics Society, which you can read in their July Newsletter (page 28).

Image credit: Envel Kerdaffrec

 

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.

GARNet workshop in Tweets

Comments: No Comments
Published on: November 29, 2012

The GARNet Tools and Technologies to Advance Plant Research workshop took place on Monday this week. It was my first attempt at live-tweeting, so I failed to take any meaningful notes for a proper blog post about it. However, you can see the abstract book here and the speakers’ slides will be online soon. In the meantime, I made a Storify of tweets from the workshop – who needs notes when you have Twitter? Thanks to @plantscience, @LiverpoolPlants, and @NeilHall_uk who all tweeted on the day and whose tweets are in the Storify.

Tools and Technologies to Advance Plant Research

Categories: methods, Workshops
Comments: 1 Comment
Published on: August 17, 2012

Next generation sequencing is now this-generation – it is the go-to method of analysis in much of molecular biology. GARNet is running a free Genetics Society Arabidopsis Sectional Interest Group workshop on the varied applications of next generation sequencing, including  identifying novel mutations, RNA sequencing and chromatin mapping, to introduce researchers new to NGS to this new technology and how it can advance plant research.

For more information, including the programme and registration form, go to:

http://www2.warwick.ac.uk/fac/sci/lifesci/news/newtech/

 

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