we need to compare multiple sequences among several strains. It's a coding region in diploid organisms. A few individuals are coming up heterozygous, with very clean sequences but many positions with ambiguous bases.
Have you tried PHRED/PHRAP. It's pretty old school and I think you need a UNIX set up as it runs in 'C'.....if memory serves!! Quality scores are assigned to each base and I've seen good alignments on some fairly shoddy sequence so if you have clean traces surrounding the SNPs the alignment will work fine. I think it was originally developed for shotgun sequencing so is more than capable of multiple alignments across the same region.
thank you!. I'll take a look at it. Softwares run with the ambiguous nucleotides... I don't know how they consider the ambiguous bases though, if they run across all possible combinations to compare 2 pairs of sequences, and how do they weight them.
I don't know if this will answer your question as It's a (very) long time since I used the software so I have no detailed recollection of setting up the algorithm parameters but dependant on the word length (I think!!!!!), and as long as the variations are limited to SNP or a few bp the alignment works fine (ambiguous bases represented as 'N'). A manual peruse of the traces is then required to identify the heterozygous pair.