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I've encountered an interesting situation with Bowtie outputting SAM format files. I'm working with some data from an Illumina run. After aligning the data using bowtie 0.11.3 and outputting the results into SAM format, I noticed that any of the reads that align to multiple sections of the genome are identified as unique reads in the SAM file (read 1 has bitwise flag value 99 and read has bitwise flag value 147). Taking the sequence and blatting it results in reads that align in multiple locations on the mouse reference.
Here are the input reads, my bowtie statement and the subsequence SAM output file:
Read 1
@I095:1:1:27:799#0/1
CATTCTCATGCCAAAGCATTGCTGCTTCCATCACACTTGGAAGTCGTCTGTTTTGCTCAACTCTTTTATTTCTACT
+I095:1:1:27:799#0/1
a^aab``XZQ[ZSRWK\__]Wa_Xa_Y]]T^a`_Z_YVJJNULX_UZ_TDL^]`VX\VNX]``a`a`TZ^^^[RSY
Read 2
@I095:1:1:27:799#0/2
GTTGGCATACTGGGAACCAGTCTCTTCATTTTCCCTATGCATGTTTTTTTTTTCCATCCTTCCTATTCTCCTATCT
+I095:1:1:27:799#0/2
aa^MO]\_a`YMMOZ`ab_a\aabaa_]_]TF\`_]TVFX]a___aaZFV_^`Z___aa__ba`U_^^\[[ZRZ\[
Bowtie command:
bowtie <mouse_reference> --solexa1.3-quals -1 s_3_1_sequence.txt -2 s_3_2_sequence.txt -S test.sam
The SAM file result is as follows:
I095:1:1:32:909#0/1 99 chr13 25412668 255 76M = 25412818 226 ATAAAATACCTTGGCGTGACTCTAACTAAGGAAGTGAAAGATCTGTATGATAAGAACTTCAAGTCTCTGAAGAAAG aaaaaa`V\^YVSN_RXLY`abaaa`XYTKMX]UZPP[QNT^a`XZ`[KZ^^OFLY\[^``RW]abb_BBBBBBBB XA:i:1 MD:Z:8T44A22 NM:i:2
I095:1:1:32:909#0/2 147 chr13 25412818 255 76M = 25412668 -226 GCTATCTTGCCAAAAGCAATCTACAGATTCAATGCAATTCCCATAAAAATTCCAAGTCAATTCTTCAACGAATTAG BBBBBBBBBBB_a`aa`a_\NYVMW^[OVDW```V_^ZRFUFYVG^_`a\T[aa\HXaba]XQWVO^]Q_aa]_ba XA:i:0 MD:Z:38C5C31 NM:i:2
Has anyone else encountered an issue where bowtie is returning a read in the repeat region but reporting it as unique?
Here are the input reads, my bowtie statement and the subsequence SAM output file:
Read 1
@I095:1:1:27:799#0/1
CATTCTCATGCCAAAGCATTGCTGCTTCCATCACACTTGGAAGTCGTCTGTTTTGCTCAACTCTTTTATTTCTACT
+I095:1:1:27:799#0/1
a^aab``XZQ[ZSRWK\__]Wa_Xa_Y]]T^a`_Z_YVJJNULX_UZ_TDL^]`VX\VNX]``a`a`TZ^^^[RSY
Read 2
@I095:1:1:27:799#0/2
GTTGGCATACTGGGAACCAGTCTCTTCATTTTCCCTATGCATGTTTTTTTTTTCCATCCTTCCTATTCTCCTATCT
+I095:1:1:27:799#0/2
aa^MO]\_a`YMMOZ`ab_a\aabaa_]_]TF\`_]TVFX]a___aaZFV_^`Z___aa__ba`U_^^\[[ZRZ\[
Bowtie command:
bowtie <mouse_reference> --solexa1.3-quals -1 s_3_1_sequence.txt -2 s_3_2_sequence.txt -S test.sam
The SAM file result is as follows:
I095:1:1:32:909#0/1 99 chr13 25412668 255 76M = 25412818 226 ATAAAATACCTTGGCGTGACTCTAACTAAGGAAGTGAAAGATCTGTATGATAAGAACTTCAAGTCTCTGAAGAAAG aaaaaa`V\^YVSN_RXLY`abaaa`XYTKMX]UZPP[QNT^a`XZ`[KZ^^OFLY\[^``RW]abb_BBBBBBBB XA:i:1 MD:Z:8T44A22 NM:i:2
I095:1:1:32:909#0/2 147 chr13 25412818 255 76M = 25412668 -226 GCTATCTTGCCAAAAGCAATCTACAGATTCAATGCAATTCCCATAAAAATTCCAAGTCAATTCTTCAACGAATTAG BBBBBBBBBBB_a`aa`a_\NYVMW^[OVDW```V_^ZRFUFYVG^_`a\T[aa\HXaba]XQWVO^]Q_aa]_ba XA:i:0 MD:Z:38C5C31 NM:i:2
Has anyone else encountered an issue where bowtie is returning a read in the repeat region but reporting it as unique?