**0**wrote:

Hello I am trying to use bowtie2 to map paired end reads that we suspect to have orignated from circular RNAs.

We are using a custom reference genome that contains only the exons from dm3.

Using BLAT on a specific read I observe that both mates map to two ends of a specific exon (this makes sense as we suspect the read to originate from a circular RNA).

However when running bowtie2 we get the following output:

```
1 reads; of these:
1 (100.00%) were paired; of these:
1 (100.00%) aligned concordantly 0 times
0 (0.00%) aligned concordantly exactly 1 time
0 (0.00%) aligned concordantly >1 times
----
1 pairs aligned concordantly 0 times; of these:
0 (0.00%) aligned discordantly 1 time
----
1 pairs aligned 0 times concordantly or discordantly; of these:
2 mates make up the pairs; of these:
0 (0.00%) aligned 0 times
0 (0.00%) aligned exactly 1 time
2 (100.00%) aligned >1 times
1100.00% overall alignment rate
1 113 chrX|"NM_001258798";|970 2005 1 20M chrX|"NM_206775";|971 2976 0 GGTTGCGCTCGTTGGAACCG IIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:20 YT:Z:UP
1 369 chrX|"NM_001201739";|969 4260 1 20M chrX|"NM_206775";|971 2976 0 GGTTGCGCTCGTTGGAACCG IIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:20 YT:Z:UP
**1 369 chrX|"NM_167594";|968 9655 1 20M chrX|"NM_206775";|971 2976 0 GGTTGCGCTCGTTGGAACCG IIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:20 YT:Z:UP**
1 369 chrX|"NM_001272735";|972 1052 1 20M chrX|"NM_206775";|971 2976 0 GGTTGCGCTCGTTGGAACCG IIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:20 YT:Z:UP
1 369 chrX|"NM_206775";|971 2002 1 20M = 2976 0 GGTTGCGCTCGTTGGAACCG IIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:20 YT:Z:UP
1 177 chrX|"NM_206775";|971 2976 1 45M chrX|"NM_001258798";|970 2005 0 GTTGTTGCTGATATATAGTGCATAGGGCAGGAAGGCGATTAAACG iIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:45 YT:Z:UP
1 433 chrX|"NM_001201739";|969 5478 1 45M chrX|"NM_001258798";|970 2005 0 GTTGTTGCTGATATATAGTGCATAGGGCAGGAAGGCGATTAAACG iIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:45 YT:Z:UP
1 433 chrX|"NM_001258798";|970 2979 1 45M = 2005 0 GTTGTTGCTGATATATAGTGCATAGGGCAGGAAGGCGATTAAACG iIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:45 YT:Z:UP
1 433 chrX|"NM_001272735";|972 2270 1 45M chrX|"NM_001258798";|970 2005 0 GTTGTTGCTGATATATAGTGCATAGGGCAGGAAGGCGATTAAACG iIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:45 YT:Z:UP
**1 433 chrX|"NM_167594";|968 10873 1 45M chrX|"NM_001258798";|970 2005 0 GTTGTTGCTGATATATAGTGCATAGGGCAGGAAGGCGATTAAACG iIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIIII AS:i:0 XS:i:0 XN:i:0 XM:i:0 XO:i:0 XG:i:0 NM:i:0 MD:Z:45 YT:Z:UP**
```

We tried modifiyng the allowed insert size using the -X parameter but this still did not yield a pair alignment.

From this thread I understand that it is possible that paired reads might not map as a pair even though both mates map if bowtie maps the two mates to different loci, but in this case both mates map to the same loci (shown in bold, which is also the gene found by BLAT) . I suspect that bowtie does not report this as an alignment of the pair because this is not the primary alignment of neither of the mates.

Is it possible to have bowtie report this as a pair alignment

thanks in advace

From what I see, it looks like you are trying to map only one read, but you should also map its mate... what command did you use ? For paired-end data, it should looks like :

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