How to get started analysing 454 data

[lindseyjane]

I have got some data from a 454 titanium sequencing run (single end) of a model plant genome and the sequencing centre have provided us with the following files:

sff/library1. sff library2.sff
mapping/ lots of files in this folder e.g. 454ReadStatus.txt

I do not have any user guide to tell me what an sff file is or what any of the files contain. Does anyone know of a good resource to get me started please?

Thanks for your help

[rglover]

Hiya,

If you're new to 454, I'd ask your sequencing centre to send you the .fna (effectively a FastA file) and .qual (the quality scores for each base) files that are produced when the .sff file is processed with the 454 software. I think that's probably the easiest way to get started

Cheers,
Rachel

[BaCh]

If you're new to 454, I'd ask your sequencing centre to send you the .fna (effectively a FastA file) and .qual (the quality scores for each base) files that are produced when the .sff file is processed with the 454 software. I think that's probably the easiest way to get started

There's a quicker way: extract all the sequences from the SFF yourself. If you don't have the sff tools from Roche/454 (which I presume you don't as they're not freely distributed), use sff_extract http://bioinf.comav.upv.es/sff_extract/index.html from Jose.

B.

[maubp]

If you don't have the sff tools from Roche/454 (which I presume you don't as they're not freely distributed), use ...

It is also worth asking your sequencing center if they can give you a copy of the Roche Newbler tools:

http://seqanswers.com/forums/showthread.php?t=114

[lindseyjane]

Thank you for these pieces of advice, I will try all of these ...

[gstitan]

I have used two types of files: sff and fasta. But results are different, is it normal?
Best is sff or fasta file??

Thanks

[maubp]

I have used two types of files: sff and fasta. But results are different, is it normal?
Best is sff or fasta file??

Thanks

SFF files contain the sequence, quality scores, adaptor clipping and flow information.

FASTA files just contain sequence information (and sometimes mixed case is used to show adaptor clipping).

If you are using Newbler, use the SFF file. There is no other way to provide the Roche 454 flow information.

[TheLight]

SFF is better because it contains the quality information about your samples. FASTA files are more popular and smaller (and therefore easy to work with).
To process your SFF files (graphic visualization, conversion, etc) you can use SFF Workbench. The bad news is that the tool is for Windows. The good news is that it has a very nice and easy to use graphic interface.

It is actually the only tool that allows you to visualize your samples (quality scores, end trimming, etc) right from the SFF file with a single click.