Hi, this is embarrassing. As a postdoc I did PCR, etc., used our department’s sequencing facility, got a surprising set of results. Two recessive temperature-sensitive alleles in the same locus, from different rounds of EMS mutagenesis screening, each had TWO missense mutations. A different pair in each allele. What are the chances?? The lesions are identified in the Materials & Methods section, which includes the following text: “Sequences… were compared with published wild-ype sequences…” Haha, speaking of missense, right? Years later, I’m thinking I should have questioned the results, done new PCRs, sequenced again. Now I’m out of the research game, have no physical lab, but I want to publish a little more on one of those mutants, a new synthetic phenotype I found years later. This might be the best opportunity to check those facts and correct them if needed. I’ve read that one can get a whole Ce genome sequence for ten bucks nowadays. Where? Or would it make more sense to go with Sanger sequencing for this? Many thanks to anyone who can advise.
I’m not sure where to get a whole Ce genome for $10… that seems a little optimistic! But if you can amplify your genomic locus with standard PCR primers, then using Plasmidsaurus for direct Nanopore sequencing of the PCR product is a very cheap ($15) and easy option that will give you ~500 raw reads, which you can directly examine for the mutations you’re interested in.
Snug
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I think Morgan is referencing Guangshuo Ou’s recent papes using Tn5 to do cost effective sequencing. I think you in theory can get it down to that price but you’d need many samples. I second using Nanopore sequencing from Plasmidsaurus etc.!
As noted, you can sequence PCR product with Plasmidsaurus for $15, just like people sequence whole plasmids.
But your concern is about the ts alleles? But if I recall it was a screen for ts alleles, right? So that part is not odd. And you are concerned about the two lesions in your favorite gene for each mutant, but that is not too unusual. Finding it twice is odd, but you note that the lesions are not the same between mutants. Was there other evidence? Rescue? These days one sequences the whole genome and then knocks in the relevant mutations to see if they are causative. But rescue plus two sequenced alleles is sufficient.
Unclear whether the sequence was compared to the database or to your own sequencing runs on the wild type. I’d prefer the latter, but not that big a deal.
Not sure I see the problem. It is right to be skeptical but sometimes a coincidence is just a coincidence
I think $10 might be in the ballpark for the sequencing costs for short-read sequencing (I haven’t looked recently, and that would have been low, but it was not wildly off) but there’s also the cost of library construction. If you pay for someone else to do it it’s a LOT more than the sequencing costs. Or you can do it yourself, and it’s not hard and the reagents aren’t terribly expensive, but last time I looked they were only sold in bulk. It’s also possible to make them almost for free, in theory, but our efforts using (other people’s) homebrew Tn5 weren’t great.
But also I’m looking at this only from my own personal experience using the facilities I had access to and the services it made sense for me to contract, a broader survey could find all sorts of things I’m unaware of.
Thanks to y’all for the speedy good answers: problem solvable. And thanks to davereiner for the optimistic note - indeed I remember feeling wonder not skepticism when those results were fresh. Be pretty cool if it were real. Now I’m having fun seeing in the alphaFold prediction that all four of those substitutions are in terra incognita, at least one of them in a disordered loop as I recall… And yes ts: in fact I recently found that one of the alleles is doubly ts: shows the LOF phenotype at both too-warm and too-cool temps - I wonder if such a thing has ever been known? That’s another question. Maybe one substitution loosens things, and the other one tightens things.
PS * correction: 'wild-ype" is not a missense, it’s a deletion. Errors about errors…
wild-hype, wild-aype… Thanx again to y’all for answers.
