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 05-13-2015, 03:00 AM #1 vinumanikandan Junior Member   Location: cochin Join Date: Feb 2011 Posts: 4 views Bowtie2 : aligned result read count not matching with sam Read count bowtie2 -f -N 1 -p 8 -x REF.fa -1 sample01_1.fasta -2 sample01_2.fasta -S sample01.sam --al sample01_al.fasta --al-conc sample01_alcon.fasta 9260783 reads; of these: 9260783 (100.00%) were paired; of these: 9260311 (99.99%) aligned concordantly 0 times 472 (0.01%) aligned concordantly exactly 1 time 0 (0.00%) aligned concordantly >1 times ---- 9260311 pairs aligned concordantly 0 times; of these: 43 (0.00%) aligned discordantly 1 time ---- 9260268 pairs aligned 0 times concordantly or discordantly; of these: 18520536 mates make up the pairs; of these: 18520180 (100.00%) aligned 0 times 356 (0.00%) aligned exactly 1 time 0 (0.00%) aligned >1 times 0.01% overall alignment rate Result: Number of reads in sample01_alcon.1.fasta: 472 Number of reads in sample01_alcon.2.fasta: 472 Number of reads in sample01_al.fasta:0 To find number of reads matching a Particular Reference from sam file I run following : perl -ne ' if (/^\@SQ/) { @F = split(/\t|:/, \$_); print \$F[2]."\n" } ' sample01.sam > agi_list.txt perl -ne ' chomp(\$_); print \$_."\t".`grep -c "\t\$_" sample01.sam ` ' agi_list.txt >1a.counts I have Couple of questions: 1) So In theory I should get (2*aligned concordantly)+(2*discordantly) =(2*472)+(2*43)=1030 . Right? but I got another number:1742 which is like (2*472)+(2*43)+(2*356)=1742. 2) I could not find the 43 aligned discordantly sequences and the 356 sequences in my alinged file why is it so and how I could find it? 3) What I need to do is calculate RPKM and also retrieve those number of sequences What I need to do is calculate RPKM and also retrieve those number of sequences Last edited by vinumanikandan; 05-13-2015 at 03:05 AM.