DEGseq and expression of novel small RNAs

[mgolo]

Hi all!

I´m new to the NGS business, and right now i have a lot of doubts about DE analysis.

I have RNA-sequenced a bacterial transcriptome in 2 growth conditions, and I have 3 biological replicates for each condition:

Condition A : Replicate 1A, Replicate 2A, Replicate 3A
Condition B : Replicate 1B, Replicate 2B, Replicate 3B

I have the bam an pileup files for each replicate.

Now, my aim is compare the expression of non-annotated non-coding RNAs in my conditions A and B (so i will use a custom annotation file).

I have read about DEGseq and i would like to use it for my DE analysis. But i have a number of questions about it:

1. What method would suit my analysis best? I have thought of using MARS...

2. How do I normalize my replicates? Should i use loess or median? What´s the difference between them?

3. What is better: to pool the 3 replicates of each condition or to analyze DE without pooling them?

4. Since my transcripts are not annotated i will have to use expression values based on raw read counts, right? Can i use the rawCount argument with the DEGseq function or is it only valid with the DEGexp function? If i use the MARS method is it automatically set to analyze raw counts?

Thanks in advance for your help!

Maria