Question: Tophat: Proper-Paired Reads
0
gravatar for ai
6.0 years ago by
ai0
ai0 wrote:

Hi Everyone,

I tried Tophat v 2.0.6 and GSNAP v 2013-02-05 to align Illumina paired-end reads. However, the numbers of proper-paired reads were very different. I also used cufflinks for assembly, but got similar number of genes/isoforms. Is there anything wrong with my commands? Why did Tophat result in such low rates of proper-paired reads? Thanks!

To get proper-paired reads: samtools view -f 0x2 accepted_hits.bam | cut -f1 | sort | uniq | wc -l

Tophat comands:

tophat -G genes.gtf -o tophat --no-novel-juncs genome read_1 read_2

Results: 1,431,500 (6.34%) proper-paired reads; 3746 genes (cufflinks).

GSNAP command

gsnap -A sam -N 0 -D ~/Software/gmap-2013-02-05/ -d mm10 -s mm10.splicesites.iit read_1 read_2

Results: 20,543,759 (90.9%) proper-paired reads;3959 genes (cufflinks).

tophat • 1.9k views
ADD COMMENTlink modified 6.0 years ago by Ashutosh Pandey11k • written 6.0 years ago by ai0
0
gravatar for swbarnes2
6.0 years ago by
swbarnes25.3k
United States
swbarnes25.3k wrote:

Isn't cut f1 | uniq going to compress two properly paired reads into a single read name?

What does samtools flagstat accepted_hits.bam tell you is the # of properly paired reads?

ADD COMMENTlink written 6.0 years ago by swbarnes25.3k
0
gravatar for Ashutosh Pandey
6.0 years ago by
Philadelphia
Ashutosh Pandey11k wrote:

Well this is not an answer but you should try '-c' to count. For e.g. samtools view -c -f 0x2 accepted_hits.bam. It should give you the counts of the properly paired mapped reads.

ADD COMMENTlink written 6.0 years ago by Ashutosh Pandey11k
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