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
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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
I think the more important question is, why don't 99.99% of your reads align at all? Before this is fixed, I wouldn't bother about any further analyses.
That is because currently I am looking into a single gene of a whole data set.
That alone shouldn't cause this.
The input file is a metagenome sequences in fasta format.