Hi all,
First forgive me if my question is trivial. I have an Illumina rna-seq dataset of two samples (the fastq files).
I have mapped them using tophat and the result of mapping so satisfactory, like nearly 70% of the fragments are mapped and then I used cuffdiff in order to know just the differentially expressed genes like below:
cuffdiff -c 0 -o output -p 12 -u /Genes/genes.gtf -L F,M sample1/accepted_hits.bam sample2/accepted_hits.bam
But the problem is that there are too few significant differentially expressed genes in the gene_exp.diff result file and I know that the correct number is really more than this.
according to my search through the net, some people believe it might be due to the inconsistency between the quality value version of my fastq flies and tophat version I have used. But I do not know how should I check it or can it be the real reason of this strange result or not?
I have used tophat 1.4.0 and this is the quality value I have gained by applying "ShortRead"R package on my fastq file:
> fastqs <- readFastq("sample1.fastq")
> qualities <- quality (fastqs)
> head(qualities)
class: FastqQuality
quality:
A BStringSet instance of length 6
width seq
[1] 46 B@@BABA5!.68A<9>67:<.43540+7(7B%%%%%%%%%%%%%%%
[2] 46 BCBA8<8@A5==9::A61?70;B=((/0:?)5:&0(:=5%%%%%%%
[3] 46 A6AA7ABBBBAA;B=B%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
[4] 46 BAA=>AABB=:BAA>>>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
[5] 46 BCBBBBA>62@B<0ABBA798-=2?82@?B%%%%%%%%%%%%%%%%
[6] 46 BA@0@/,:B<AA;A7B=<3&5B59?.%%%%%%%%%%%%%%%%%%%%
Any suggestion would be appreciated.
First forgive me if my question is trivial. I have an Illumina rna-seq dataset of two samples (the fastq files).
I have mapped them using tophat and the result of mapping so satisfactory, like nearly 70% of the fragments are mapped and then I used cuffdiff in order to know just the differentially expressed genes like below:
cuffdiff -c 0 -o output -p 12 -u /Genes/genes.gtf -L F,M sample1/accepted_hits.bam sample2/accepted_hits.bam
But the problem is that there are too few significant differentially expressed genes in the gene_exp.diff result file and I know that the correct number is really more than this.
according to my search through the net, some people believe it might be due to the inconsistency between the quality value version of my fastq flies and tophat version I have used. But I do not know how should I check it or can it be the real reason of this strange result or not?
I have used tophat 1.4.0 and this is the quality value I have gained by applying "ShortRead"R package on my fastq file:
> fastqs <- readFastq("sample1.fastq")
> qualities <- quality (fastqs)
> head(qualities)
class: FastqQuality
quality:
A BStringSet instance of length 6
width seq
[1] 46 B@@BABA5!.68A<9>67:<.43540+7(7B%%%%%%%%%%%%%%%
[2] 46 BCBA8<8@A5==9::A61?70;B=((/0:?)5:&0(:=5%%%%%%%
[3] 46 A6AA7ABBBBAA;B=B%%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
[4] 46 BAA=>AABB=:BAA>>>%%%%%%%%%%%%%%%%%%%%%%%%%%%%%
[5] 46 BCBBBBA>62@B<0ABBA798-=2?82@?B%%%%%%%%%%%%%%%%
[6] 46 BA@0@/,:B<AA;A7B=<3&5B59?.%%%%%%%%%%%%%%%%%%%%
Any suggestion would be appreciated.
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