Optimal Analysis Software?

[sjhanson]

I am carrying out an RNA-seq project in which I hope to identify DE genes between two conditions in a non-model organism. I have two biological and two technical replicates for each condition, with 75bp Illumina reads. My reference sequence is a low-coverage (1-2X), unannotated genomic sequence obtained by 454 GS FLX titanium sequencing. This reference sequence has been assembled, and consists of ~140,000 contigs/singletons that range from a few hundred base pairs to a few kb in length.

My questions are about the best software to use for my analysis. From my own literature search, it seems like most mapping software will work, but in terms of DE analysis, I'm not very sure. For example, it appears that DEGseq requires an annotated genome. Can the parameters be adjusted so that annotation is not required? Does any other DE software require annotations? Would it somehow be possible to specify the contigs/singletons as "genes" and simply examine DE among them (and subsequently identify which genes/gene fragments are contained in the sequence)?

It is also not clear to me which software can deal with biological and technical replicates most effectively. Which programs can and cannot account for these?

I am new to NGS and relatively new to bioinformatics, so I apologize if my questions are somewhat naive. Any help would be appreciated!

[helene_thygesen]

personally I like Vencio's method, described in

http://www.biomedcentral.com/1471-2105/5/119/

It was sevelopped for SAGE but it works fine with RNAseq.

The R code can be downloaded from the journal.

Alternative, you can use edgeR.