Antarctic nematode conundrum!

[tryptophan]

Hey guys, this is my first post! Sorry if i got the wrong forum for this question but i wasn't sure where i should stick it.

Our lecturer at uni posed us students with an interesting question for an assignment. The assignment is based on the antarctic nematode P. davidi, which can tolerate freezing temperatures. He said we have to discuss ways we can determine exactly how this little critter can tolerate freezing using high-throughput sequencing techniques. The transcriptome of a close relative to P. davidi has been determined.

I was thinking of using 454 pyrosequencing to start off with due to P. davidi not being sequenced before (i.e., not having a reference sequence??), and using this in conjunction with a different sequencing type, perhaps Illumina Solexa which has greater accuracy - especially over homopolymer dNTP repeat sections.

How would the use of these sequencing techniques shed light onto how this nematode tolerates freezing? Could sequencing of the genome find specific genes that are only found in this nematodes genome perhaps?

Any help on this topic would be much appreciated!!

Sincerely,
Confused student.

[Thorondor]

well it all depends on how much time and money you can spend on this project. ;-)

you could try to sequence the transcriptome (mRNA) first, with deep sequencing only using Illumina with a close relative as reference this might work out fine. Check for expression rates and transcripts that are not in the close relative (cheapest and fastest way).

De novo sequencing of the whole genome takes lot more time and is lot more complicated and of course more expensive.

Anyway you should first check if their are known genes that are responsible for this freezing toleration and if so you already know what you are looking for.

[HESmith]

I suspect that your professor is less interested in what technology you would use and more interested in how you would identify candidates responsible for freezing tolerance once the sequence is known. You should focus on the latter issue.

[tryptophan]

Thanks for the replies guys! Hmm good idea to try sequencing the transcriptome first. Would that involve making a cDNA library with all the data you have at the end of the sequencing method?

Research suggests that at least some of the freezing tolerance of P. davidi comes from proteins (IAPs - ice-active proteins). But despite "considerable work", it has not proved possible to identify the proteins responsible for freezing tolerance.

SO i'm thinking that if you could find transcripts unique to P. davidi (i.e., transcripts not in the close relative), you could perform site-directed mutagenesis on these coding regions and see the effect on the whole organism...maybe. Or alternatively isolate the IAPs produced when the organism is stressed with decreasing temperature and use mass spec to find the sequence, find it on the transcriptome and THEN used site-directed mutagenesis. Is that even possible or am i being far-fetched?

Thanks for reading this by the way, i love talking about questions in a forum environment and bouncing ideas around, it really helps my understanding!

[PHSchi]

And how would you introduce your mutated sequences into an organism where no genetic tools are available, that is even parthenogenetic?
If you were looking for a molecular tool, RNAi seems the only that might work at all. However, that would bear some major challenges in this organism and the life stages you are looking at.
No this can be done with NextGen, but you do not need a cDNA library but several!

[tryptophan]

The strain of P. davidi that we would be experimenting on is parthenogenetic. Wouldn't you be able to introduce mutations into the sequence after you obtain the sequence via nextgen techniques??

[PHSchi]

You can introduce mutations into any sequence that you know (regardless if you know it from Sanger or Second Gen.), it's as 'simple as doing PCR and cloning' (well, okay, there is a lot of fiddling and tweaking as always). However, if I understand you right, you do like to bring the mutated sequence back into the organism and that is a big problem in your system!

[HESmith]

It should be illuminating to review the literature and see how next-gen sequencing has been useful in other model systems to characterize gene function.

[tryptophan]

Indeed HESmith, i have read multiple papers on characterizing freeze tolerance in a few other nematodes, and even a freeze tolerant frog, and the methodologies in these have been very insightful.

[HESmith]

Please read my last message again; I didn't say anything about freeze tolerance.

[tryptophan]

Oh, i'm sorry. I misread your post.