The command samtools fastq will directly convert a bam file to fastq files. Combining with the filter parameter, we could output unmapped read pairs by -f 12. 0x0004 in the flag field of the SAM format means “the query sequence itself is unmapped” and 0x0008 in the flag field of the SAM format means “the mate is unmapped”. Therefore, 0x0004 + 0x0008 or 0x0004 | 0x0008, i.e. -f 12 means both the query sequence itself and its mate are unmapped.

samtools fastq -f 12 -n -1 out.R1.fq -2 out.R2.fq in.bam

References

1. http://www.htslib.org/doc/sam.html

2. http://www.htslib.org/doc/samtools.html