After going through the IUPAC nucleotide code list, everything now makes sense. It shows that the ambiguous positions could be mutations or not Mutations. Now the purpose of my analysis is to identify SNPs relative to reference sequences.
1)How do I determine whether or not these ambiguous nucleotide positions are mutations or not?
2) which is the best approach to check these SNPs?
If you have the chromatogram files from your Sanger sequencing, you should manually check the chromatograms for both strands of your template. If the nucleotides at specific positions of interest are still ambiguous, it would be advisable to re-sequence your target region.
Hi,
If you are familiar with the R programming environment, you might use the package "sangerseqR" from BioConductor for more-or-less automated analysis. From the Sanger seq (ab1 chromatogram file) you can get primary sequence (which is your main fasta basically), but you can also get a secondary sequence, which show you the alternative base per position (the next most probable one by the peak intensity). You can define the minimum intensity ratio of the primary and secondary peaks to select the probable SNPs positions.
Anyway, if you have rather small area it still might be easier to look through manually.
Best,
Michail
The answer depends on what type of organism you are studying and what steps you took prior to sequencing. For diploid eukaryotic organisms and uncloned nuclear genes you would be expecting a 50:50 ratio of two bases at a site where the individual being sequenced is heterozygous. But for mitochondrial genes, bacteria, viruses, tetraploid plants, fungi in their monoploid stage, and other organisms you would not be expecting a 50:50 ratio. Any amplification method such as PCR can also throw the ratio off.
However, the peak height on Sanger sequencing chromatograms is rarely indicative of the ratio of bases actually present in the sample. More often the peak heights are variable due to not equal incorporation of bases in different contexts by the polymerase being used. So you should not be expecting a perfect 50:50 peak height ratio even if you know for sure your sequenced DNA was a diploid heterozygote.
I think the easier and faster way to check the SNPS is analyzing your amplicon in both senses and checking the electropherograms manually, and if you want to be completly sure, you must make a new PCR amplification (if thats the case) and sequencing it again. And if it is a SNP you will see both peaks wit the same height
To be really thorough, and to phase the SNPs, you can clone and sequence your PCR products. You'll have to sequence a large number so you have a good chance of getting all of the different versions.
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