I used bowtie2 to do the alignments.

Do you think that the methodology described below is correct?

1) To extract the reads that align only once

 grep -E "@|NM:" alignments.sam |  grep -v "XS:" > unique.sam

Now I am looking for a way to find which reads' alignments don't overlap with any other alignment.

2) Converting to bam & sorting:

samtools view -Sb unique.sam > unique.bam
samtools sort unique.bam -o unique.sorted.bam

3) Then view the sorted file:

 samtools view GCF_000090985.2_k15_local_verySensitive.unique.sorted.bam | head -n 4

sl157419384     0       NC_013038.1     67349   22      35S30M10S       *       0       0       ACCCCTTTGCCCAGNTCCCCTTTGCCCAGNNCCCCTTTGCCCCCTCCCCCTTTGCCCCNTTCCCCTTTGCCCCNT     IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII        AS:i:57 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:23C6       YT:Z:UU
sl157419385     0       NC_013038.1     67349   22      20S30M25S       *       0       0       TCCCCTTTGCCCAGNNCCCCTTTGCCCCCTCCCCCTTTGCCCCNTTCCCCTTTGCCCCNTGCCCCTTTGCCCNGA     IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII        AS:i:57 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:23C6       YT:Z:UU
sl157419386     0       NC_013038.1     67349   22      5S30M40S        *       0       0       NCCCCTTTGCCCCCTCCCCCTTTGCCCCNTTCCCCTTTGCCCCNTGCCCCTTTGCCCNGATCCCCTTTGCCCNGA     IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII        AS:i:57 XN:i:0  XM:i:1  XO:i:0  XG:i:0  NM:i:1  MD:Z:23C6       YT:Z:UU
sl158880407     16      NC_013038.1     180163  22      22S9M3I40M1S    *       0       0       NCCAGCCGAGGAGGTAGCAGCCGAGGAGGNTAGAGCCGAGGAGGNCNGAGCCGAGGAGGAGGGAGCCGAGGAGGN     IIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII        AS:i:59 XN:i:0  XM:i:5  XO:i:1  XG:i:3  NM:i:8  MD:Z:7G11A0G0G16T10     YT:Z:UU

...

I think that the 4-th column represents the starting position of the alignment on the genome.

4) Since my reads have constant length, I intend to iterate all over these starting positions, add the length and compare with the next line to see if any overlapping happened.

What do you think about these steps? Do they make sense, or I am missing something?

Thanks