Hi I'm new to SEQanswers, and fairly new to bioinformatics, but I'm trying to work with the python scripts for the DEXSeq R package through Bioconductor.
We have paired-end data from human samples, (4 controls 4 treatments), using SOLID. I have access to the BAM files, that were aligned using TopHat (hg19).
Whenever I get the text output I have nothing but 0 values for all the count values. I was able to work through some of the example data, but am having no luck with our actual data.
Here are the steps I've taken (and admittedly I'm using the code(s) provided with the packages). I personally don't do a lot of programming but am actively learning, including from many resources I've found browsing the forums on SEQAnswers.
-Went to http://useast.ensembl.org/info/data/ftp/index.html and obtained the gtf file for humans, and put it in a folder with the python scripts dexseq_prepare annotation.py & dexseq_count.py, and the human bam files.
In bash terminal:
- gunzip Homo_sapiens.GRCh37.67.gtf.gz
- python dexseq_prepare_annotation.py Homo_sapiens.GRCh37.67.gtf Homo_sapiens.GRCh37.67_DEXSeq.gff
- samtools index human1.bam
- samtools view human1.bam > human1.sam
- sort -k 1,1 -k2,2n human1.sam > human1_sorted.sam
- python dexseq_count.py -p yes Homo_sapiens.GRCh37.67_DEXSeq.gff human1_sorted.sam human1_dexseq.txt
The above executes, and I get a .txt file, but always gets 0 values for all of our samples. I originally thought it might be that we were using SOLID data; however, I also tried with some human Illumina data and had the same end result. Because the only entry not containing the a 0 value is the "empty" I'm thinking it is a problem I have either with the gtf/gff file or more likely not sorting the SAM file properly. Is the above correct? Or am I missing something? Also does the gff file need sorted as well?
Any help is much appreciated. Thank you. Oh and if it helps I'm working on a Mac OS X 10.7.4.
We have paired-end data from human samples, (4 controls 4 treatments), using SOLID. I have access to the BAM files, that were aligned using TopHat (hg19).
Whenever I get the text output I have nothing but 0 values for all the count values. I was able to work through some of the example data, but am having no luck with our actual data.
Here are the steps I've taken (and admittedly I'm using the code(s) provided with the packages). I personally don't do a lot of programming but am actively learning, including from many resources I've found browsing the forums on SEQAnswers.
-Went to http://useast.ensembl.org/info/data/ftp/index.html and obtained the gtf file for humans, and put it in a folder with the python scripts dexseq_prepare annotation.py & dexseq_count.py, and the human bam files.
In bash terminal:
- gunzip Homo_sapiens.GRCh37.67.gtf.gz
- python dexseq_prepare_annotation.py Homo_sapiens.GRCh37.67.gtf Homo_sapiens.GRCh37.67_DEXSeq.gff
- samtools index human1.bam
- samtools view human1.bam > human1.sam
- sort -k 1,1 -k2,2n human1.sam > human1_sorted.sam
- python dexseq_count.py -p yes Homo_sapiens.GRCh37.67_DEXSeq.gff human1_sorted.sam human1_dexseq.txt
The above executes, and I get a .txt file, but always gets 0 values for all of our samples. I originally thought it might be that we were using SOLID data; however, I also tried with some human Illumina data and had the same end result. Because the only entry not containing the a 0 value is the "empty" I'm thinking it is a problem I have either with the gtf/gff file or more likely not sorting the SAM file properly. Is the above correct? Or am I missing something? Also does the gff file need sorted as well?
Any help is much appreciated. Thank you. Oh and if it helps I'm working on a Mac OS X 10.7.4.