I performed an RNA-seq experiment with Illumina paired-end sequencing. I’m working with Pseudomonas aeruginosa and uploaded the genome of interest (fasta) and gene annotation file (gtf) file from pseudomonas.com which is a database for all pseudomonas. Those 2 files are from the same strain.
When performing FastQC, the webpage tells me the data is in Sanger / Illumina 1.9. All reads are of excellent quality, with a phred score mean of 39.
I am using Bowtie2 for alignement and then Htseqcount for counting the reads. When using bowtie, i put the 2 sets of read in the paired-end option. The tool works but then when i put it in Htseq count, the summary of the output is this:
| Category | Bowtie2 on data 55 | data 1 | and data 2: alignments |
|---|---|---|---|
| __no_feature | 29798440 | ||
| __ambiguous | 0 | ||
| __too_low_aQual | 887230 | ||
| __not_aligned | 2034538 | ||
| __alignment_not_unique | 0 | ||
| __aligned |
It is as if there is nothing that was aligned with Bowtie2?
I performed another analysis with Rockhopper and everything seems to map correctly. I am very confused as to why nothing is aligned in the summary.
If anybody has any idea as to why it doesn’t work, it would help tremendously. Thank you.
