Hi-

This is my first time using a bioconductor package.
I also have never worked with genetic data before. Here is a list of my
DEXSeq issues thus far. Just places within the April 4th and Feb 27th
vignettes that I haven't succeeded in replicating for my own data using
 module r/3.0.1 within a bash shell.

Info: I used tophat with a fastq file, and then was able to to create a sam
file.

Issue #1

module load htseq/0.5.3p9
python
import HTSeq
alignment_file = HTSeq.SAM_Reader(" sam file ")

This step generates a SAM alignment object.

However, I need to exit in order to use PBS code to run the
dexseq_prepare_annotation.py
and dexseq_count.py and generate gff and txt files,
and thus lose the SAM alignment object which I am guessing should
be used as the gtf file?

Can I actually skip the alignment_file step if I already posses a gtf file?

Issue #2

If I have a homo sapiens transcript.bam file (not from ensembl) how do
I process the file in order to make it work with
dexseq_prepare_annotation.py?

Should I pass the transcript.sam file through the alignment_file code first?

Issue #3

Using an ensembl animal gene data, I have to specify strandedness=no within
the
python dexseq_count.py code and I am wondering if this will
lead to errors down the line.. The dexseq_count code won't work without
-s no, despite that the bam file is probably from the ensembl site.


Issue #4

The two vignettes use different ways to create an ExonCountSet object.I am
confused about which to use...

Also, if I have a homo sapiens gene transcript data from illumina, put
through tophat,  what type of functions should I use to prep the data for
the ExonCountSet
creation? Same question for ensembl animal gene data...

Should I prep the data by using this workflow:
http://www.bioconductor.org/packages/release/bioc/vignettes/GenomicFeatures/inst/doc/GenomicFeatures.pdf

Thanks,
ML
ASU Grad Student

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