Question: Primer design from bam file ?
0
gravatar for Picasa
10 months ago by
Picasa470
Picasa470 wrote:

Hi Biostars,

I have 5 samples that I mapped against a genome.

My goal is to design set of primers (final amplicon size around 100bp) from this, that will allow to amplify regions to look for SNPs between individuals.

Do you know a software/method that take bam files, looking for conserved regions with SNPs inside (that are present in my 5 samples) and produce set a primers ?

Thanks a lot for your help.

primer map • 396 views
ADD COMMENTlink modified 10 months ago by WouterDeCoster40k • written 10 months ago by Picasa470

Can you tell the purpose of primers ?

ADD REPLYlink written 10 months ago by k.kathirvel93200

To amplify regions and looking for SNPs between individuals

ADD REPLYlink written 10 months ago by Picasa470
1

You should probably just call SNPs in the samples and then see which SNPs could distinguish/identify these individuals. Not everything has to (or should be) automated, of course. Once you know the SNPs that distinguish/identify them, you can then design primers accordingly.

I wrote a SOP for designing primers a few years ago, from which you may get a few tips: Designing a single set of primers and probe for a genomic region of interest

ADD REPLYlink modified 10 months ago • written 10 months ago by Kevin Blighe48k

Just a suggestion: First, create consensus sequence from bam files for each sample (Generate Consensus sequence from BAM file ). Design primers to your reference genome sequence (Primer3, https://www.ncbi.nlm.nih.gov/tools/primer-blast/ ), and then mach them to your consensuses, to see if they mach correctly (Primer Map (http://www.bioinformatics.org/sms2/primer_map.html ). Best, Agata

ADD REPLYlink modified 10 months ago • written 10 months ago by agata88790

Hey Agata, please check your URLs / links. There is a bug in the system whereby, if a parenthesis appears after a URL, it is interpreted as part of it. You just need to add a space between these.

ADD REPLYlink written 10 months ago by Kevin Blighe48k
1

Corrected, thanks! Agata

ADD REPLYlink written 10 months ago by agata88790

Thanks Agata, but not sure to understand what the consensus sequence is ?

ADD REPLYlink written 10 months ago by Picasa470

Consensus sequence is a linear sequence from bam file (mapped reads). For example, if you have sample reads mapped to reference genome, you can generate the linear representation of sequence for this sample. It will include all differ variants (SNPs).

ADD REPLYlink modified 10 months ago • written 10 months ago by agata88790

In case you don't want all SNPs, but only the unique for each sample, you can compare consensus linear sequences from 5 samples, and then design primers in desire places. Agata

ADD REPLYlink modified 10 months ago • written 10 months ago by agata88790

Suggestion: if by "mapping file" you mean "bam file" then just write the latter. Explicit is better than implicit. I have adapted your title.

ADD REPLYlink written 10 months ago by WouterDeCoster40k
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