Dear community,
I used subread-align to map RNA-seq reads to the reference genome (after index building) resulting in SAM files. Now, I am additionally interested in the unmapped reads (not aligning to the reference), but the subread-package seems to only show the number of unmapped reads and is not collecting them.

Questions:
1. Is there a way to collect unmapped reads within the subread-package
2. Otherwise, can you suggest a program able to do this?

Thanks a lot

The SAM files from Subread contain all the reads, both mapped and unmapped. You can use standard samtools utiliites to extract any subset of reads you want, e.g., mapped or unmapped reads. It should be easy to figure out how to do that from a Google search or from the samtools manual.

I don't know about subread-align but outside BioC, the STAR aligner offers the option --outReadsUnmapped Fastx that will dump the unmapped reads to FASTQ files.

cheers,

robert.

As Gordon said the Subread output includes both mapped and unmapped reads. It is fairly straightforward to extract unmapped reads from a SAM file. The 'RNAME' column (the 3rd column) in the SAM file contains a value '*' for each unmapped read.