Mothur and fastq file format

[elizondas]

Hi all.

I am completly new at NGS. I have just got back my datas from a first run of 454 pyrosequencing. We want to get into metagenomics and we have decided to use mothur.
Before to start with mothur, I'd like to have a look to my results using FastQC. The problem is that I don't have the fastq format, just the sff, fna and qual. I would know how to do that on linux, but we are using windows terminal. Does anybody now how to extract the file on windows? sff_extract doesn't work.

By the way I would like to know which one would be the next step we must follow, I mean, from my demultiplexed samples. I guess it would be the denoising step, but not sure....

Thanks for the help,

Elisa

[Brian Bushnell]

BBTools can change fasta+qual to fastq, and runs in Windows.

Unzip and untar it (7-zip can do this) to some directory, for example C:\BBMap\

Then you would run:

java -ea -Xmx200m -cp C:\BBMap\current\ jgi.ReformatReads in=reads.fna qfin=reads.qual out=reads.fastq

Let me know if you have any trouble!

[elizondas]

It didn't work
I am working in the directory in which I have the files, and I get a message error saying :
Exception in thread "main" java.lang.RuntimeException: can't find file 454Reads...then, there are more messages....

[Brian Bushnell]

Could you give the exact command line you used and the complete error message?

[cliffbeall]

One thing about FastQC is that it is geared towards analyzing high complexity sequences, so that that amplicons (I assume that's what you're doing) will likely cause warnings about overrepresented sequences. It will work ok for length, quality, etc. - though mothur also can tell you about those in a less fancy format.

[maubp]

Does anybody now how to extract the file on windows? sff_extract doesn't work.

I am surprised, sff_extract is written in Python and so ought to work under Windows. Did you get an error message? What exactly was the command you ran?