454 CWF files

[joa_ds]

Hi fellow sequence analysts.

I am working on a 454 basecalling project and I have 2 main questions/problems.

1) It really would help me if someone could tell me a method to get the controlbead sequences into the SFF format/file. I am suspecting Roche filters on keysequences before exporting them to the SFF file. But they must be present in the raw CWF files, so they must be exportable to SFF format. I'll buy some beers if you have a method.

2) Is anyone aware of a Perl module/function that allows binary CWF files to be accessed. Having access - through perl - to the raw intensity would help me forward quite a bit. I am currently using a colleague of mine's R package, but I am not quite the R wizard. So I have to go through the CWF to txt via R, access txt via perl method to get things done.... Priority is @ problem 1 though

[flxlex]

1) It really would help me if someone could tell me a method to get the controlbead sequences into the SFF format/file. I am suspecting Roche filters on keysequences before exporting them to the SFF file. But they must be present in the raw CWF files, so they must be exportable to SFF format. I'll buy some beers if you have a method

I looked into if this was possible using the 454 image analysis software, but no luck (unless one has the source code)...

[joa_ds]

Well,

Back in the old days (like 1 year ago or so...) the basecaller produces two sets of fasta files. TCA.fa and ATG.fa if I remember correct. One file was the real file, the other one with controlbead sequences.

I guess it was the Titanium upgrade that stopped exporting the control bead sequences file. It must be some little setting in the basecaller software... Having the source would be so handy indeed...

[kmcarr]

Well,

Back in the old days (like 1 year ago or so...) the basecaller produces two sets of fasta files. TCA.fa and ATG.fa if I remember correct. One file was the real file, the other one with controlbead sequences.

I guess it was the Titanium upgrade that stopped exporting the control bead sequences file. It must be some little setting in the basecaller software... Having the source would be so handy indeed...

It is still entirely possible to create those FASTA files for the control reads today using the gsReporter application.

# gsReporter --fna=control <my.cwf>

This will create two files, my.ATG.454Reads.fna and my.CAT.454Reads.fna, one file each for the two types of control beads. The possible arguments to --fna are library (default), control or all. You can produce the corresponding quality files by replacing --fna with --qual.

You had originally asked about getting the control reads into the SFF file; that does not appear possible. The SFF files are created by the gsRunProcessor and it is not possible to configure what it writes to the file. While the gsReporter application help text indicates that it has an --sff output function, it states not to use it as it is only maintained for backward compatibility. Indeed when you try to use it the program simply states:

gsReporter: The --sff option is not available in this version of the gsReporter.