[Kashliks]

Hey austic

I almost wanted to make new thread about SNP filtering parameters, but I might as well join this.
I am also using Lasergene package for evaluation.

According to publications there must be around 20-25k SNP in human exome. Not sure about variability in these numbers between individuals, but I suppose thats target.

So I set parameters to:
show All SNPs,
show Coding SNPs only (as I am interested in exome),
Q call 40,
P not ref - 90%,
and SNP percent filter 50-100.

Last one is most arguable because it says to show SNPs that have been seen in at least half (50%) of reads - I think its too stringent, but it is only way that allows me to arrive at 23k SNPs (which supposedly are expected) and gives me transition/transversion (Ti/Tv) ratio of 3.05 (exome is supposed to be 3-3.5, random is 0.5 and genome on average is 2.0-2.1) if I relax any of parameters above, then this "check" fails.

Any thoughts and comments on these parameters?

Now there is toughest part - to narrow down in order to find rare syndrom cause.
I saw in this forum scheme about narrowing pipeline numbers, but there were no explanation how it was done (like 20k->10k->700->120 and then 4-6).
Would be nice if someone would share info how to get from 23k to at least 1000 or less so its possible to look at them manually


Some more points about my data: I am surprised that there is about half of SNPs that are not annotated (isn't that too many NOVEL?), also half are supposed to be amino acid change snps (also too many for what i know about genetics), 169 STOP codon SNPs - OK supposedly possible.

I am thinking of looking at all trio at once in order to catch new SNPs in child - most likely fakes.

Anyone knows how to check SNP conservation in evolution (I mean to check 23k at once) and same for amino acid change SNP (11k at once in polyphen and/or similar soft).

Would be nice to share filtering parameters and numbers what comes out - so its possible to compare.

[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.

[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]

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

[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]

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]

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]

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)

[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]

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!

[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]

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

[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!!

[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]

anyone?? please...