Separating aligned reads

[thh32]

I have been using bowtie2 to align my reads to an indexed reference genome and used the --un-conc and --al-conc functions to separate out the aligned and non aligned reads into separate files, however I have just received a new data set which has been merged. I have tried using the -U option in bowtie2 to see if this worked but it doesn't seem to. Does anyone know of another program I could use for this and the appropriate command? I am looking into BWA to see if this is an option but am new to the program.

Thanks,
Tom

[Brian Bushnell]

You can do that with BBMap:

(index)
bbmap.sh ref=reference.fasta -Xmx29g

(map)
bbmap.sh in=reads.fq outm=mapped.fq outu=unmapped.fq -Xmx29g

That will split it into two fastq files. If you change the output file extensions to ".sam" instead of ".fq", you will get sam output. If you have paired reads in 2 files, you would do this:

bbmap.sh in1=reads1.fq in2=reads2.fq outm1=mapped.fq outm2=mapped2.fq outu1=unmapped1.fq outu2=unmapped2.fq -Xmx29g

The "-Xmx29g" indicates how much memory java is allowed to use; set that at ~85% of your physical memory.

[swbarnes2]

samtools view can filter your .bam for mapped or unmapped reads. Unmapped reads will have the 4 flag set, mapped reads will not.

[thh32]

@Brian Bushnell I have fasta files, would BBMap still work this this?

@swbarnes2 I dont have a .bam file so cannot use samtools

Bowtie now accepts my files but is just outputting into the .sam file instead of the --un-con or --al-conc files I have specified although the files them-selves are created by BOWTIE2

[sphil]

doesn't --un-con and --al-conc just state wether pairs could be mapped concordantly and not whether they could be mapped at all?

[thh32]

@sphil yes it does, just realised this and without -conc i works absolutely fine. Thanks for all your help.

[Brian Bushnell]

@Brian Bushnell I have fasta files, would BBMap still work this this?

Yes, BBMap works with fastq or fasta files.