DNASTAR SeqMan NGen3

[duck808]

Hello all, I'm new to this site and trying to learn how to use SeqMan NGen3 from DNASTAR.
I was wondering if anyone is familiar with this program, because I need to learn how to use this program as soon as possible for my research.

I have tried it once, but it does not show any graphs. For example, in this site http://www.dnastar.com/skins/skin_1/...luminaRef.html
it showed some graphs after the assembly, but I didn't get any of that. Also, I used different formats during the process (e.g. .ab1 and .sff formats).

Anyone know which process went wrong?

Thank you.

[duck808]

anyone?? please...

[ECO]

Patience. 15 hours is not enough of it. I'm going to go ahead and guess that you could call the company and ask them?

[duck808]

Patience. 15 hours is not enough of it. I'm going to go ahead and guess that you could call the company and ask them?

I did contact the company, but I was just wondering if anyone else on the forum knows how to use this program.
hahaha, you are right.... I need patients.

Thanks!!

[austic]

re: DNASTAR SeqMan NGen

Hi duck,
I have also just picked up this software and given it a go. I guess I would ask what system/platform you are running the SeqMan NGen software on - if running on WinXP(i86), then the SeqMan Pro contig analysis module is supposed to automatically start when the SeqMan NGen assembler is done running and input the assembly automatically as well. However I did see a note in the help manual that you may have to manually open the assembly (.sqd file) in SeqMan Pro - as I have to do because I have to run SeqMan NGen on a 64-bit Linux OS as my Windows OS is only 32-bit

[austic]

once you get SeqMan Pro up and running, double-click on a contig to get a graphical view, or right-click for the different view options

[austic]

As an only marginally related post, I have a question for the community regarding contig building in general - I am working on reference-guided whole genome assembly for a bacteria (the little bugger has 2 chromosomes and ~7.1Mbp DNA in it's genome) using DNASTAR's SeqMan NGen 3.0 and I am in an interesting spot in that I can get the assembler to read my raw data (.sff data from a 454 machine) and assemble contigs, but I get a LOT of contigs out in the end - often >1,000. and that is not helpful when it comes to the analysis as i am interested in SNP's and other small mutations which are listed by contig - it would also be helpful if I could view genes and other genetic elements in relation to my SNP's during my data analysis and to do that I will need the contigs to align to the annotated genome, which they apparently do not want to do (?) - I can't seem to figure it out, anyway

So my question for the community is this: what assembly parameters should I change first to make the contig assembler construct longer contigs, and which parameters should I avoid at the risk of introducing major errors (I don't care about how long it will take or how much space it will take or any other consideration) - I am a total n00b at all this sequence analysis stuff - never even took a class in undergrad. so any input (or resources) would be a great help.
thx!

[duck808]

hey austic thanks for the reply. maybe we can help out each other, would you send me an email to [email protected] ?

thanks.

[austic]

problem fixed - was using the wrong file type as a template and the software was defaulting to a de novo assembly (hint. .gb not recognized as .gbk - my bad)

[neha]

Seq Man Ngen DNASTAR

Hello Everyone,

I am new to using DNASTAR. I have downloaded it and installed it properly. I want to do the genome assembly for my reads for E.coli generated by Ion Torrent.

I have seen the video available on the site and tried to do the same for my reads but getting an error that assembly was not successful.

input reads : e.coli.sff
template : e.coli.fasta

Any help would be appreciated

Thanks,

Neha

[austic]

my suggestion would be to download the .gbk file for e.coli and try that as the template, I tried using a different file format for mine and it wouldn't assemble properly either

if you still have trouble,try opening the .gbk file in seqBuilder and outputting the genome as the dnastar file format (.seq?)

[neha]

Thanks Austic. I tried the way you have suggested but still I am not able to do assembly. I am still getting the same erroe that assembly was not successful. I dont know if there is something wrong with my reads or is it solely template problem.
I am taking the .sff file generated from Ion torrent as reads. I also converted the .gbk file into .seq format through Seqbuilder but again that is of no help.

[austic]

sorry I couldn't help you, neha. (also sorry for the delayed response - things are finally quieting down here after the holidays)

[neha]

Analyzing DNASTAR report

Hey Austic,

No issues. Finally, I have done De novo Assembly for one of the Vibrio cholerae strain using DNASTAR. I have following questions regarding the analysis

a) I am getting so many contigs more than 1000 and almost all of them are repeat regions. How to filter out these repeat regions and will this affect the assembly.
b) There is difference in the coverage of real contigs and repeat regions.
c) What parameters should I use for calling SNP as it calls SNP even on 1x coverage.

Actually this is my first assembly using this software so getting confused in the interpretation of the results.

Thanks in advance

Neha

[austic]

Neha,
I'm sorry, I'm afraid I have no experience using the de novo assembly. I know that you can contact DNASTAR and they will schedule a training session (webinar) for you where they walk you through the process, hopefully they can help you with the assembly portion.

as far as filtering SNPs, yes the DNASTAR software is not set to filter any SNPs on by default, the DNASTAR rep I spoke with encouraged using the following parameters for filtering SNPs: a depth of 15 reads (although I'm sure this will depend on the technology used to generate the data), a SNP% of about 90-100%, and completely ignoring the Indels. Although my data was generated on a Roche 454 platform and that particular technology has difficulty with homopolymeric repeat regions so there is usually an abundance of Indels (that are not actually present in the sample). There are fields at the top of the SNP report for filtering SNPs in SeqMan.