Easy way to assemble sequences from a plasmid insert
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2.7 years ago
gbl1 ▴ 80

Hi all,

I have done some old fashion cloning and sequencing of about 3000pb with for each of them. I have got one side opposite side and one or two internal reads.

my text editor is not doing it well and I'm sure someone designed something better!

What is actually the best way of having an assembled sequence? Is there any on-line tool I could just stuff in my reads and having direct answer?

Thanks in advance

Benjamin

sanger Assembly plasmid clone • 776 views
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You could give DNAbaser a try (trial available). Many commercial programs (if you have access) like Vector NTI ContigExpress, Sequencher, DNASTAR should all be able to do this as well.

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I use SnapGene to view all my sequencing (the Viewer is free, and has the ability to align sequences still in the free version I think). I beleive you can also use Phred/Phrap/Consed.

From a quick google, the assembler CAP3 is capable of doing this too.

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Thanks for your answer.

CAP3 is a very bad advise for that… I use it for other purpose, but here it won't work: A sanger sequence has usually a end that is really approximate, so cap 3 won't recognize it.

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your reply puzzles me a bit, why wouldn't CAP3 work here? It was especially developed for assembling Sanger sequences (as back in the days we did not have any other sequencing).

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Basically, I have two sequences that should be assemble on 3', 3' sequences are not accuarate in sequencing, therefor, Cap3 does not assemble them for the ends to assemble does not look the same for it.

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OK, that I understand but still ... perhaps some parameter tweaking might help to resolve this?

in any way, if the similarity between the ends is very low practically all assemblers will have problems to assemble them.

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I think what you mean in that case is that your data isn't very good, such that CAP3 is failing, not that CAP3 isn't suitable for this purpose, which is otherwise a bit of a misleading statement.

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Assembling 2 Sanger sequences (of a given clone) is my purpose… End of Sanger sequence is not good enough for CAP3… that's it… Cap 3 is good for many things, but not that one…

PS: I precise for those who haven't get it, of course, after, I will get an assemble sequence, that CAP3 would easilly deal with!

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2.7 years ago
colindaven ★ 3.2k

Ugene - downloadable - not online as requested, but a very useful tool. Can even handle NGS reads.

https://ugene.net/wiki/display/UUOUM15/Molecular+Cloning+in+silico

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I download it and try… indeed, it seems a very interesting tool in many aspect… But could you tell me how it could be useful for the given question?

(I actually solve my issue the hard way, but I'm going to have some more to do, so if there's a way to do it quickly and safely, I would appreciate)

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It gives you interactive access to the CAP3 assembler as mentioned above, and also allows for explorative read mapping - so it's a lot better tool than a text editor.

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Oh… as mentioned above CAP3 is not suitable for that…

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