How to detect Heterozygous (double) peaks from Sanger

[shuang]

How to detect heterozygous (double) peaks from a Sanger sequence?

I found an article (http://seqcore.brcf.med.umich.edu/do...interpret.html) suggest Sequencher, Mutation Surveyor, and polyphred can detect heterozygous bases. But I haven't had luck in applying any of them to approach my purpose.

Any one has experiences in using any software for detecting heterozygous bases from a Sanger sequence?

[Michael.James.Clark]

I just throw them in FinchTV and look with my eyes. I guess that might be a lot of work if you're doing more than a handful of samples, though.

[shuang]

I have hundreds of Sanger sequences. Thus, I'm seeking a tool which can process samples by an automatic method.

[Michael.James.Clark]

I have hundreds of Sanger sequences. Thus, I'm seeking a tool which can process samples by an automatic method.

Oh, well in that case, I know exactly which tool you could use: An undergrad.

[pmiguel]

How to detect heterozygous (double) peaks from a Sanger sequence?

I found an article (http://seqcore.brcf.med.umich.edu/do...interpret.html) suggest Sequencher, Mutation Surveyor, and polyphred can detect heterozygous bases. But I haven't had luck in applying any of them to approach my purpose.

Any one has experiences in using any software for detecting heterozygous bases from a Sanger sequence?

I have used polyphred. This was years ago and even then it seemed antiquated. What sort of failure are you seeing? Is the software not detecting the hets that you verify exist visually?

At hundreds of samples, wouldn't you be better using 454 amplicons?

--
Phillip

[maubp]

When you say 100s of Sanger reads, are you wanting to look for heterozygous (double) peaks within the individual chromatograms? Or would it suffice to align the called bases and hope about half show each variant (much like SNP detection with NGS data)?

[shuang]

Yes, I need a software which is able to detect heterozygous (double) peaks within individual chromatograms. So, when performing SNP discovery next, those individuals would be recognized as heterozygotes.

I actually is using a trial of CodonCode Aligner, which is based on Polyphred. It seems working. However, it doesn't let me import any large reference sequences.

If I use Polyphred, can I input a 735Mb genome sequence or at least a whole chromosome as a reference sequence?

Why I use Sanger? ... not decided by me.

[pmiguel]

If I use Polyphred, can I input a 735Mb genome sequence or at least a whole chromosome as a reference sequence?

Probably not.

Why do you need a reference? Can't you just assemble de novo? I thought that was what I did when I used polyphred long ago. I remember looking at the results in consed.

--
Phillip

[shuang]

Because that would be useful to know a genome location of each SNP.

[krobison]

I haven't looked at the various products carefully, but I suspect few if any with Sanger support have been updated for the post-genome era. The way I hacked this at my previous employer was to generate my own Genbank files with the region of interest AND the region encoded in the filename. We had Mutation Surveyor already, which I used to generate the calls. Their mutation tables could then be written in simple tab-delimited form & a final program converted the positions back to genome-wide coordinates.

A hack, yes. But it worked.

[trackavinash]

Hi Shuang,
Just curious, did you finally accomplish this ? I'm trying to call het SNPs from individual chromatograms but Polyphred seems to assemble multiple reads and then create a consensus contig.
Thanks!

Yes, I need a software which is able to detect heterozygous (double) peaks within individual chromatograms. So, when performing SNP discovery next, those individuals would be recognized as heterozygotes.

I actually is using a trial of CodonCode Aligner, which is based on Polyphred. It seems working. However, it doesn't let me import any large reference sequences.

If I use Polyphred, can I input a 735Mb genome sequence or at least a whole chromosome as a reference sequence?

Why I use Sanger? ... not decided by me.