Multiple hits in bowtie2 alignment

Question

[znq0928]

Hi, I am new to the RNA-Seq. I have a problem that when I map the reads using bowtie2, over 80% of the reads were aligned > 1 times, only around 10% were aligned exactly 1 time, which is the opposite as I expected. I am wondering if there is anything wrong with my data or I did anything wrong while using bowtie2? Thanks!

[JC]

what is your data? did you run some QC analysis before doing alignment? why are you using bowtie2 if this is RNAseq?

[znq0928]

Hi, my data was RNA seq reads from illumina NextSeq system. I did run QC analysis and trimmed the data with trimgalore. I use bowtie2 to map reads to the genome and then I can use htseq-count to count reads that mapped to each gene. After that I'll use DESeq2 to do differential gene expression analysis. Is there anything wrong with the pipeline?

[JC]

1. mapping to the genome is only ok for prokaryotic genomes
2. after trimming, how was your average size? multi-mapping problems generally are because reads were too short and align in multiple positions
3. after aligning, are you checking where is aligning? looks like you have a bad library (rRNA garbage)
4. other tools are aware of splicing and many RNA-seq artifacts (HTSeq2, STAR, Salmon, ...)

[znq0928]

Thank you very much for your answer! 1. My target organism is a bacteria, so should be fine 2. I set the cutoff as 75 bp and quality score as 28, so should be roughly 75 ( I did a single end 75 bp run) 3. No I didn't check the aligning position, but I do aware there is round 20-30% rRNA contamination after the rRNA removal. 4. I run htseq-count after the alignment, there are many read that were thrown to the too_low_aQual which I though it should be ambiguous or alignment_not_unique. What do you think that might come from? Plus, the genome of the bacteria I'm working on is only 3Mb, and I got 15-20M reads, so do you think the multiple hits in bowtie2 is caused from the sequencing depth? Thanks again for your help!