Reverse compliment VERY different to forward sequence! - (May/19/2008 )

Hi,

I should start off by telling you that Im a newbie to sequencing and phylogenetics so speak S L O W L Y!!

I am currently trying to reconstruct the phylogeny of the Translation Elongation Factor 1 alpha for 180 isolates of a pathogenic fungus. I have amplified using the primers EF-1 and EF-2 (see jpeg solid arrows) and then sequenced with the forward primer (EF-1). The forward sequencing reaction failed for around 30 samples and so I sequenced the reverse (EF-2). I am now trying to align the sequences (with clustal) but the reverse compliment sequences cluster together and are very different to my forward and reference sequences. I am not sure what to do and why the EF-2 reverse compliment sequences would be so different? Any suggestions would be a great help.

hello relf,
you say you sequenced forward and reverse, this might have created sequences that won't be identical, you might need to create contigs this means that you'll get an overlap between your fwd and rev sequences, then a larger sequence.

xxxxxxxxxxxxxxxxxxxxxxxxxxxx this representing your fwd
this would be the rev xxxxxxxxxxxxxxxxxxxxxxxxxxxx

the contig would be
xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx

got it? to create contigs i think there are tools online, otherwise you can use phredPhrap (google consed). phred is a base caller (it means it identifies the bases that appear in your chromatogram) and phrap creates the contigs.

i hope this is useful to your ends. cheers.