Question: Easy way to assemble sequences from a plasmid insert
0
gravatar for gbl1
8 months ago by
gbl170
gbl170 wrote:

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

plasmid assembly clone sanger • 328 views
ADD COMMENTlink modified 7 months ago by colindaven1.7k • written 8 months ago by gbl170

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.

ADD REPLYlink written 8 months ago by genomax71k

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.

ADD REPLYlink modified 8 months ago • written 8 months ago by Joe14k

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.

ADD REPLYlink written 8 months ago by gbl170
1

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).

ADD REPLYlink written 7 months ago by lieven.sterck5.9k

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.

ADD REPLYlink written 7 months ago by gbl170

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.

ADD REPLYlink written 7 months ago by lieven.sterck5.9k

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.

ADD REPLYlink written 7 months ago by Joe14k

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!

ADD REPLYlink modified 7 months ago • written 7 months ago by gbl170
0
gravatar for colindaven
7 months ago by
colindaven1.7k
Hannover Medical School
colindaven1.7k wrote:

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

ADD COMMENTlink written 7 months ago by colindaven1.7k

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)

ADD REPLYlink written 7 months ago by gbl170

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.

ADD REPLYlink written 7 months ago by colindaven1.7k

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

ADD REPLYlink written 7 months ago by gbl170
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