sam flag is confusing

[poorphd]

i mapped reads to my transcrpitome assemblied from those reads.
there is a record in the sam file
HWI-ST397_0000:5:1101:19602:2158#ATCTCG 20 UN57620 131 25 95M *
0 0 GTTGATCTGAGAGTAGCAGATGCCCTCAATCTTCACATCCTTGGGCACTTTGGCGCCCATGTCA
GTGTCAGAGGGCTGGATGCTCGTGAAGGTGC BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB
BBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBBB XT:A:U NM:i:5 X0:i:1 X1:i:0 X
M:i:5 XO:i:0 XG:i:0 MD:Z:11C3C11G63C1T1


i think the flag=20, = 16+4, 16 means reverse, 4 means unmapped, but i found it can mapped from 131 on UN57620

the mapped sequences are here, not reverse
>UN057620
CCCGCTATAGCCATGATCCTAGCTTGAAAATCTCGTTGCG
GCAAAATTTATGCTTTGTCGGCGGATAAAGGAGGGTAAGT
GTATGTTTTGTTTACAAAGAGGAATCAAATCAGTGTAGGA
CGACAGTCTAGTTGATCTGAGCGTACCAGATGCCCTCGAT #UN057620
GTTGATCTGAGAGTAGCAGATGCCCTCAAT #read
CTTCACATCCTTGGGCACTTTGGCGCCCATGTCAGTGTCA #UN057620
GAGGGCTGGATGCTCGTGAA#read
CTTCACATCCTTGGGCACTTTGGCGCCCATGTCAGTGTCA #UN057620
GAGGGCTGGATGCTCGTGAAGGTGC#read

[ETHANol]

Agree the sam flag is very confusing and I am thoroughly confused by it. The sam file documentation doesn't help.

Can someone explain it in plain English.

What is the flag for reads that map uniquely? Can I sort these out with a awk/sed/grep?

[swbarnes2]

The flags as such don't say if it mapped uniquely. bwa adds the XT tag. If it says XT:Z:U, that means it mapped uniquely. Ifit says XT:Z:R, then that end was repetative, but that doesn't mean tha the mate didn't map uniquely.

The simplest explanation for that tag is that you are doing single end reads, with bwa, and this read hangs off of one reference sequence, and onto another. bwa concatenates sequences together, and when one read crosses two sequences like that, it will map them and give a 4 in the flag.

[nilshomer]

See: http://picard.sourceforge.net/explain-flags.html

[yina]

Nilshomer, thanks a lot, this app is very usefull, but I still don´t understand the logic behind the flag system...

I have some alignemnts with flag = 0 , can someone explain this??

[swbarnes2]

Nilshomer, thanks a lot, this app is very usefull, but I still don´t understand the logic behind the flag system...

I have some alignemnts with flag = 0 , can someone explain this??

Single end reads yield only 3 possible flag values: 0,4 and 16.

0 means the read aligned in the forward direction. 16 mean it aligned in the reverse direction. 4 means it didn't align.

The rest of the flags are for paired end data only.

[yina]

Thanks swbarnes2, I found a good explanation of the way the bitwise flag is constructed, here the link:

http://seqanswers.com/forums/showthread.php?t=2301&highlight=bitwise+flag

Hope it helps ETHANol and others