Not consistent result between SAM filtering and bowtie

Question

[Picasa]

Hello,

I have a list of contaminants that I want to filter out from my paired end:

bowtie2 -x contaminants -1 pair1.fastq -2 pair2.fastq -S out.sam

Now I want to extract only unmapped reads from the .sam file but I want to keep only paired end.

So if read1 map somewhere and read2 not, I want to discard these PE.

samtools view -f 4 -bu input.bam | samtools view -f 8 -bu - | java -jar  picard-tools/picard.jar  SamToFastq  I=/dev/stdin F=out1.fq.gz F2=out2.fq.gz  FU=unpaired.fq.gz

I got a problem, the output of bowtie 2 is :

5317475 reads; of these:
15317475 (100.00%) were paired; of these:
15155323 (98.94%) aligned concordantly 0 times
155208 (1.01%) aligned concordantly exactly 1 time
6944 (0.05%) aligned concordantly >1 times
----
15155323 pairs aligned concordantly 0 times; of these:
95593 (0.63%) aligned discordantly 1 time
----
15059730 pairs aligned 0 times concordantly or discordantly; of these:
30119460 mates make up the pairs; of these:
29853788 (99.12%) aligned 0 times
195884 (0.65%) aligned exactly 1 time
69788 (0.23%) aligned >1 times
2.55% overall alignment rate

which mean I should have 15155323 PE right ?

But when I do :

grep '@' out1.fastq | wc -l

I got : 14798490 PE

1) What's wrong with my command ?

2) Moreover my unpaired.fq.gz is empty

[Pierre Lindenbaum]

Hello picasa1983!

It appears that your post has been cross-posted to another site: http://seqanswers.com/forums/showthread.php?t=70248

This is typically not recommended as it runs the risk of annoying people in both communities.

[Picasa]

Sorry but I try to increase my chance of answers. This problem annoy me and it seems like nobody got an answer.

[GenoMax]

Try read binning approach for a 1 step solution: C: BBSplit syntax for generating builds for the reference genome and how to call di