Hi all,
I performed EMS-induced mutagenesis and I want to identify the specific mutation sites.
Is there any recent, best methods to identify the mutation sites rather than complementation test?
Thank you.
Your question is extremely vague. I’ve tried to answer it helpfully, but am not entirely sure what you’re looking for.
Assuming your new mutant has a distinctive and easily scored phenotype, one that it shares with mutations in only a handful of known genes, complementation testing is going to be by far the cheapest and probably the quickest way to assign your mutation to a known gene, or to suggest it’s not an allele of a gene known to affect that phenotype and so likely to be worth pursuing further.
Mapping your mutation can also be very helpful, especially if there are too many candidate genes that mutate to similar phenotypes for you to do complementation tests with all of them, or if for some reason complementation tests aren’t going to be easy (if your mutant phenotype or that of a reference allele isn’t recessive, for example, or if the phenotype can only be scored on a population basis, which makes the complementation test a bit harder to do). Good mapping data that places your gene in an interval with a very small number of known candidate genes can suggest it’s worth the time to do relatively difficult complementation tests with alleles of those genes, or simply to sequence those genes’ coding regions in your mutant, or even to attempt transgenic rescue with cosmids or PCR products containing wild-type copies of those genes, or potentially to attempt transgenic phenocopy with a PCR product of one of those genes amplified from your mutant strain.
Whole-genome sequencing is an option, but it’s expensive, it’s relatively slow compared to a few complementation tests, and for best effect really should be in combination with complementation data or with genetic mapping. Whole-genome sequencing is likely to give you dozens of candidate genes that you can most effectively rule in or out based on mapping or complementation data.
There are a bunch of book chapters (in WormBook, for example) and papers on how you can map mutants and do complementation tests, and use newer technologies such as whole-genome sequencing. I would strongly recommend that you talk more with your adviser or find some experienced person nearby who can discuss this with you.
Our experience is somewhat different from Hillel’s. We’ve had a lot of success identifying mutations by whole-genome sequencing with the Hawaiian mapping method (see https://www.ncbi.nlm.nih.gov/pubmed/21079745). It’s quick (only one cross to the Haw strain plus picking F2 mutants) and cheap ($200/mutant strain) if you consider the cost of labor involved in other mapping/cloning methods. From 50 mutant F2s, we typically identify 1-5 non-synonymous candidate mutations, which we validate by RNAi or, more recently, CRISPR.
Dear Hae-Eun,
Indeed you can identify mutations caused by EMS using next-generation sequencing, with mapping and identification combined
(ie no mapping required beforehand). Several methods are available and are described in the Wormbook / Genetics chapter that we have recently
published (Genetics. 2016 Oct;204(2):451-474), using either back/out-crossing or a cross to the Hawaiian strain, and whole-genome sequencing.
We and others have successfully used the former, using EMS-density mapping (https://www.ncbi.nlm.nih.gov/pubmed/20610404),
which takes advantage of backcrossing to map and identify the causal mutation (while performing basic genetic tests on your mutants).
I would recommend you read this chapter to determine which approach suits your needs better.
Best luck!
These are all great comments. Cloning from EMS alleles is a complex equation. If you want specific advice, I recommend being specific in your description of what your alleles are: phenotypes, penetrance, etc. With specific information, there are a lot of very experienced worm geneticists who come here who can give you great advice.