Question: Bowtie2 mapped just 50% of the reads
0
gravatar for silas008
5 months ago by
silas008100
Brazil
silas008100 wrote:

Hey, guys. I need some help with Bowtie2. It will be great if you can help me.

I am trying to align small RNA seq data to C elegans genome. The pre-processing is ok (good base quality, no adapters, reads are trimmed and etc). But in the alignment step bowtie2 only aligned about 50% of all reads. This is not common. I have used Bowtie2 many times to make this kind of alignments and it always worked well.

The read lenth is about 22nt and the command line used to this mapping was:

bowtie2 -p 4 --very-sensitive-local -x ce11  -U trimmed.fastq -S .sam

Do you know if there are big differences between Bowtie2 Versiosn 2 and Bowtie2 Version 3. I am using version 3 for the first time.

Thank you very much.

rna-seq • 288 views
ADD COMMENTlink modified 5 months ago by Biostar ♦♦ 20 • written 5 months ago by silas008100
3

Doesn't Bowtie2 manual say that Bowtie1 performs better with reads that are shorter than 50 nt? Your reads are barely longer than the initial seed length of --very-sensitive-local

Perhaps the reads that did not align are shorter than 20 nt?

ADD REPLYlink modified 5 months ago • written 5 months ago by 5heikki8.4k

It is a good point. I will try it again with Bowtie1 to see what happen.

Thanks

ADD REPLYlink written 5 months ago by silas008100

Have you checked for contamination?

ADD REPLYlink written 5 months ago by jrj.healey13k

I think this is not the problem because STAR aligner have mapped more than 90% of the reads.

Another point is that I think I mapped this data about 1 year ago using Bowtie2.2.7 instead of the last release and it also worked well.

ADD REPLYlink written 5 months ago by silas008100

Very probably right. With low mapping rates contamination is always my first suspicion though :P

ADD REPLYlink written 5 months ago by jrj.healey13k

In general contamination, as rRNA, have millions of reads overexpressed. I have checked the overexpressed reads in fastqc and they are miRNA reads.

Do you think should I use a specific program for that?

Thanks

ADD REPLYlink written 5 months ago by silas008100

Is your library stranded?

ADD REPLYlink written 5 months ago by Macspider2.8k
1
gravatar for Mehmet
5 months ago by
Mehmet470
Japan
Mehmet470 wrote:

If you do aligment of RNAs to genome of an eukaryotic organism, C. elegans in this case, you should use splicer aligner. STAR is a splicer aligner, meaning needs exon-intron boundaries to align RNA reads to genome. Hisat2 is another one.

You should use splicer aligner for this reason. This is why you get very low mapped ratio when you used Bowtie compared to STAR.

ADD COMMENTlink written 5 months ago by Mehmet470
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