sequence assembly program - (Sep/16/2014 )
Hallo,
anyone here that knows a good (free) program to assemble some sequences?
I am using a (paid) program, but my results are a bit strange and I would like to try another one just to see whats going on.
(EDIT: others than this: http://www.ebi.ac.uk/Tools/msa/clustalw2/, so no multiple alignment programs , I should have mentioned this before.. haha)
since you cite clustalw2, can we assume you want to perform multiple sequence alignments?
if so then you can try dialign (i found this, amongst others, at expasy).
if not, then ignore this post, or, maybe you can find the software you want at expasy.
mdfenko on Tue Sep 16 11:49:28 2014 said:
ah yes, that website! I forgot about it.
I am trying to sequence a plasmid... but something very weird is happening... I have no clue what is going on, but the sequences just don't align.. Its weird, never had this problem before.
what sort of result are you getting? is the sequence running through the insert (assuming you have one)?
are you sanger sequencing or ngs?
if sanger, are you sequencing from both strands of your plasmid? (sorry if this appears insulting, it's not meant to be) did you remember to reverse complement one of the sequences?
did you run duplicates? if so, how did they compare?
mdfenko on Tue Sep 16 12:26:56 2014 said:
what sort of result are you getting? is the sequence running through the insert (assuming you have one)?
are you sanger sequencing or ngs?
if sanger, are you sequencing from both strands of your plasmid? (sorry if this appears insulting, it's not meant to be) did you remember to reverse complement one of the sequences?
did you run duplicates? if so, how did they compare?
I am getting sequences that do align (lets say sequence X aligns with sequence Y) but this result does not seem to align with sequence Z , although sequence Z has 200 base pairs in common with it...(but after those 200 base pairs, all of a sudden it does not match anymore... it seems that pieces of the plasmid have been inverted/duplicated and stuff like that..)
Its just so strange!
It seems that there is something really wrong with the plasmid!
And yes: I sequence from both sides of the plasmids.. but now I am just doing a "primer walking" approach: I just have the contigs and make new primers based on this.
But this is the weird part: I have 2 contigs. I make primers to extent the contigs (at the end) but primer 1 that is based on config 1 (to extent) all of a sudden gives a sequences that matches with an inner part of contig 2 ! Go figure...
I am not sequencing it myself, a company does it, its sanger sequencing....
And its not even about an insert! Its a plasmid I am trying to sequence.... but the results that come out of it are pretty weird! Never seen this before.
My weirdest experience was a double-sized plasmid with a mutationally inactivated origin on one of the copies. It sequenced nearly perfectly, but was the wrong size. But it cut and gave restriction fragments that were correct. I'd guess something like this is happening, with double copies of some of your fragments. This causes real confusion, since you now have two places that a primer binds to. I solved the problem by single cut enzyme, religation, and re-transformation.
phage434 on Tue Sep 16 21:32:57 2014 said:
My weirdest experience was a double-sized plasmid with a mutationally inactivated origin on one of the copies. It sequenced nearly perfectly, but was the wrong size. But it cut and gave restriction fragments that were correct. I'd guess something like this is happening, with double copies of some of your fragments. This causes real confusion, since you now have two places that a primer binds to. I solved the problem by single cut enzyme, religation, and re-transformation.
What program do you use ?
Any good online (free) one?
The multiple alignment programs are of course not what I need and I seem to find many of those, but not the "regular" ones for what I am trying to do.
you can check the software cited at this site.
mdfenko on Wed Sep 17 12:06:30 2014 said:
you can check the software cited at this site.
Ok,
I already tried some others, same crappy results... so there is, for sure, something wrong with the plasmid. It seems a region got duplicated.
Some NGS assemblers may support Sanger sequences, I think ABySS does. Maybe it's a bit overkill though.
DNA Baser is a paid program but it's fully functional during the trial (30 days) period