How to collapse forward & reverse Sanger reads into consensus sequence?

[Harmakhis]

Hi everybody,

I relatively new to this, so I thought I'd ask about way to solve my problem in the most easy way.

I have *.ab1 files from an Applied Biosystem's 3730xl DNA Analyzer from different individuals for different genes each sequenced with a forward and a reverse primer.

In the past I constructed the consensus sequences by hand in BioEdit while looking at the chromatogram in Geospiza's FinchTV but that's a lot of work and quite subjective.

I thought there must be an easier and more objective way to do this... Is there a way/workflow to automatically combine my two reads per gene and individual in a consensus sequence using some kind of objective quality score and outputting that as a FASTA file or similar? Ideally, there should be a way to flag heterozygotes, too.

Any help would be greatly appreciated.

[GenoMax]

Commercial software programs (Sequencher, Vector NTI Alignx, CLC Workbench, Geneious etc) can do this very easily but I assume you are looking for a free/open source alternative.

[Harmakhis]

You're right, an open-source/freeware program would be best. I came across SeqTrace but I have trouble getting it to work. Somehow when I input my *.ab1 files no confidence score is displayed and that stops the program from calculating a consensus sequence.

[Harmakhis]

Okay, just as information: it seems SeqTrace is doing what I want. The problem now is that it needs embedded Phred Quality Scores in the chromatogram to work.

The files I get from my sequence provider are ab1-files withOUT the quality scores. I can download the quality scores as seperate .qual files.

Is there anyway to combine those (preferably .abi or .scf files, as those are the file types SeqTrace reads)?

[maubp]

You could try a basecaller like TraceTuner to recall the bases from the chromatograms, that should give you ABI files with bases and PHRED quality scores.

[Harmakhis]

This didn't work either, and I finally know why: I got a reply from my service provider and it seems there was something wrong with the files. I got new ones and now I have the PHRED quality scores and such.

Thanks for your help!