Multiple hits in bowtie2 alignment
0
0
Entering edit mode
4.8 years ago
znq0928 • 0

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!

rna-seq • 1.0k views
ADD COMMENT
0
Entering edit mode

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

ADD REPLY
0
Entering edit mode

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?

ADD REPLY
0
Entering edit mode
  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, ...)
ADD REPLY
0
Entering edit mode

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!

ADD REPLY

Login before adding your answer.

Traffic: 1634 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6