Extracting Unmapped Reads from BAM files using Rsamtools

Question

[dk0319]

Hello,

Does anyone have any experience extracting unmapped reads from BAM files and converting them back to fastq format using Rsamtools? If you could provide some generic code to address this problem it would be greatly appreciated.

I am mapping C. elegans RNA library against E. coli genome to remove contamination. I would like to obtain the unmapped reads to then map against C. elegans genome for DGE.

PS. These are unpaired reads

Cheers

[GenoMax]

Posting an alternate solution. You can take a look at bbsplit.sh from BBMap suite. You can easily bin reads by aligning to both these genomes at the same time.

[benformatics]

You can also use GenomicAlignments to pull the unmapped reads out. You really gotta wrangle the metadata and pull all the right values to extract the necessary information to recreate the original fastq lines - but it is do-able.

Example for single end:

readGAlignments(bam.file,params=scanBamFlag(isUnmappedQuery=TRUE)

Answer 1

[Shalu Jhanwar]

You can use SAM flag value 4 to get reads unmapped from BAM files like below:

samtools view -f 4 in.bam > out.bam

You can provide this flag with Rsamtools as well.