Paired-end reads mapped 50% while each pair reads mapped 80%

[zack80.liu]

Hi all,

I am new to the RNA seq world. I have processed bunch of illumina paired end reads. The reported alignment is only 49%.. When I use bowtie to map each pair of the paired-end reads, I got 80% of the reads mapped.

bowtie -n 3 -p 10 --best -e 200 --trim5 15 --trim3 25 --sam BowtieIndexes/mm9 -1 Raw_files/sample-1_export.fq -2 sample-2_export.fq sample.sam


# reads processed: 21482797
# reads with at least one reported alignment: 10637665 (49.52%)
# reads that failed to align: 10845132 (50.48%)
Reported 10637665 paired-end alignments to 1 output stream(s)


I was wondering if this is normal. Here's the command and output. Thanks.

P.S.: This is my 3rd post on this forum. I posted a similar question in my 2nd post. However, I can no longer reply that post.


----- I couldn't post a reply, so here it goes ----

Isn't it that tophat also uses bowtie to align reads?

[frozenlyse]

You shouldn't be using bowtie for RNA-seq (especially with PE reads) - try tophat or another one of the splicing-aware aligners

[zack80.liu]

Isn't tophat also use bowtie to do the alignment?

[fkrueger]

Dear Zack,

The most likely explanation would indeed be that a sizeable fraction of you library fragments are spanning a splice junction. In these cases many fragments in your library are longer than the allowed Bowtie default fragment size of 250bp (These parameters can be adjusted with
-I/--minins <int> Default: 0.
-X/--maxins <int> Default: 250)

Using TopHat or any other splice junction aware aligner should indeed fix your problems.