Hi,

I aligned the reads to the amplicon sequence using bowtie2. Here is the summary of my run:

113624 reads; of these:
  113624 (100.00%) were paired; of these:
    2008 (1.77%) aligned concordantly 0 times
    576 (0.51%) aligned concordantly exactly 1 time
    111040 (97.73%) aligned concordantly >1 times <--
    ----
    2008 pairs aligned concordantly 0 times; of these:
      20 (1.00%) aligned discordantly 1 time
    ----
    1988 pairs aligned 0 times concordantly or discordantly; of these:
      3976 mates make up the pairs; of these:
        2445 (61.49%) aligned 0 times
        40 (1.01%) aligned exactly 1 time
        1491 (37.50%) aligned >1 times
98.92% overall alignment rate

The bowtie2 summary statistics suggest that 111040 reads aligned to multiple locations (since it says aligned concordantly >1 times) of the amplicon sequence. However, when I ran samtools view -f 0x100 sample.sorted.bam | wc -l, (where the sam flag of 0x100 suggests not primary aligned reads), the output is 0. Moreover, when I run the following command samtools flagstat sample.sorted.bam, I get the following result:

227248 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
224803 + 0 mapped (98.92% : N/A)
227248 + 0 paired in sequencing
113624 + 0 read1
113624 + 0 read2
223232 + 0 properly paired (98.23% : N/A)
223308 + 0 with itself and mate mapped
1495 + 0 singletons (0.66% : N/A)
20 + 0 with mate mapped to a different chr
2 + 0 with mate mapped to a different chr (mapQ>=5)

Can someone please explain the discrepancy? Thanks!