samtools extract unmapped reads

Question

[wrab425]

I have been given the following command to extract unmapped reads from a bam file

samtools view -b -f 141 ${SAMPLE_ID}.ecoli.bwamem.bam > ${SAMPLE_ID}.unmapped_ecoli_2.bam

1. I am unsure what the -f flag and 141 means
2. Is it necessary to sort the bam file before running this command?
3. I have consulted the samtools view manual and it is not clear why this command should extract unmapped reads.

Thanks

Answer 1

[ATpoint]

See this link that explains flags: http://broadinstitute.github.io/picard/explain-flags.html

For all unmapped reads do:

samtools view -f 4 -o ${SAMPLE_ID}.unmapped_ecoli_2.bam ${SAMPLE_ID}.ecoli.bwamem.bam

-f means keep and -F means do not keep so in the above command you keep only unmapped reads. Extraction of reads where both the read and the mate are unmapped would be -f 13 given it is paired-end.

You can then modify based on your needs with the linked tool that gives you the flags you want based on the read properties.

[wrab425]

Thanks ATpoint, that was very helpful. I am just getting rid of contaminating reads that map to ecoli before mapping to my host genome so I do not think that I need to sort. Is that correct? I think your command is doing the same thing as mine and that if I use the o flag with a bam file then I do not need a b flag and the > sign. Is that correct?

[ATpoint]

Yes, this all sounds correct.