problems closing gaps phage genome

[rexxi]

Hi there!,

I suscribe to this forum cause I notice that is really useful for everyone who is working un NGS technology.

Well, my trouble starts with de gap closing of a phage... there are just 6 contigs but the trouble is that there is not references to use to make primers or orient the postition of them.

I tried to use GapResolution software, but newbler 2.6 doesn't give me 454Scaffold.txt file althought I selected the option "output scaffolds" and if I don't have this, I can't do anything with it.

I tried to desing primers with a pair of softwares, but a contig is as little that it doesn't give primers.

I don't know what strategy to take... Please I need your help.

greetings

[kmcarr]

Hi there!,

I suscribe to this forum cause I notice that is really useful for everyone who is working un NGS technology.

Well, my trouble starts with de gap closing of a phage... there are just 6 contigs but the trouble is that there is not references to use to make primers or orient the postition of them.

I tried to use GapResolution software, but newbler 2.6 doesn't give me 454Scaffold.txt file althought I selected the option "output scaffolds" and if I don't have this, I can't do anything with it.

I tried to desing primers with a pair of softwares, but a contig is as little that it doesn't give primers.

I don't know what strategy to take... Please I need your help.

greetings

Regarding the lack of any scaffold output, did you include any paired end sequence data in your assembly? Without any paired end data newbler (or any other assembler) can't create scaffolds.

If you truly don't have a reference (a closely related phage may work in this case) to orient your contigs relative to each other you could use a multiplex PCR strategy to fill in the gaps. See the section "CLOSING GAPS BETWEEN YOUR ASSEMBLED CONTIGS" on this web page.

[rexxi]

Regarding the lack of any scaffold output, did you include any paired end sequence data in your assembly? Without any paired end data newbler (or any other assembler) can't create scaffolds.

If you truly don't have a reference (a closely related phage may work in this case) to orient your contigs relative to each other you could use a multiplex PCR strategy to fill in the gaps. See the section "CLOSING GAPS BETWEEN YOUR ASSEMBLED CONTIGS" on this web page.

I was searching for a closely related reference but I didin't find anything. And we don't have a paired end sequence data, for this I am designing primers, but as I was not as clear of the strategy to take, you idea of multiplex pcr is a real option to my proyect. Thanks you very much, best regards for you.

PD: don't you know a optimal primer designer to do this, in linux or windows? I'm trying with FastPCR 6.2 and amplifx, but I don't know if there is a better option

[flxlex]

You might want to try the contig graph file (454ContigGraph.txt) newbler generated. My guess is you have a few repeats breaking up your assembly into 6 contigs OR the genome consists of six unlinked pieces. If you look at my post explaining the file here, you may be able to figure out how the contigs are oriented relative to each other. Also, check out the contig graph visualisation program mentioned in the comments (direct link here).

[rexxi]

flxlex, I was reviewing your suggest, but I have troubles whit it because in my 454ContigGraph.txt file there is not a line with the scaffolds information (and I don't have a Saccolfs file in my output files set) I was viewing the options that I have to do this, and the option of multiplex pcr is a little tedious and I'not sure that if it really effective. For this, I'll try the primer-walking strategy to close it. I'm really glad of your disposition and your help guided to me to find some solution Thanks.