I know this question have raised many times here and in other forums but I've tried everything other people suggested in previous posts and still can't figure it out where is the problem...... This is the output summary of Bowtie2 (I checked several times the target genome, hg19 and it's correct):
21404130 reads; of these: 21404130 (100.00%) were paired; of these: 21196512 (99.03%) aligned concordantly 0 times 104527 (0.49%) aligned concordantly exactly 1 time 103091 (0.48%) aligned concordantly >1 times ---- 21196512 pairs aligned 0 times concordantly or discordantly; of these: 42393024 mates make up the pairs; of these: 906397 (2.14%) aligned 0 times 28296440 (66.75%) aligned exactly 1 time 13190187 (31.11%) aligned >1 times 97.88% overall alignment rate
I have paired-end ChIP-seq data, 50 bp reads. These are my steps (in Galaxy):
1) I groomed the fastq files to get fastqsanger (I checked if it's correct: Input FASTQ quality scores type --> Sanger & Illumina 1.8+)
2) FastQC is ok for all samples, some adapter contamination.
3) I trimmed using either TrimGalore or Trimmomatic (I get the same result using both) using forward and reverse files for each sample. I checked and I didn't missed up the forward and reverse samples (I run Bowtie2 using first file 1 and file 2 and also using first file 2 and second file 1 and I get the same result...)
4) I mapped the trimmed files to hg19 genome using Bowtie2 with option -fr and I get the result shown above. No matter what I try, I always get the same with the different samples that I have. All of them have around 32% of reads with adapters which I trimmed. I have no idea what to do with this.
Any suggestions or idea of where is the mistake? Am I missing something?
Thank you very much in advance.