[lh3]

The flag. It is the mate-unmap bit that can be mislabeled. The unmap bit is always set correctly.

[Ting Zhang]

The flag. It is the mate-unmap bit that can be mislabeled. The unmap bit is always set correctly.

Ok, but for the other best hits listed in "XA", no flags are given, so how can we judge if they are also mapped?
And sometimes, the first best hit appear in in "XA" again, but sometimes not, why? What's the difference between the first best hit and best hits in "XA"?
An example is as follows, where the "hg19_refGene_NM_001190241" appear in "XA" again:
GA-C_0001:1:1:1519:3751#0 83 hg19_refGene_NM_001190241 2021 0 49M = 1932 -138 NCTTTCTCATAAGAATGAAATCTTGGAAATTGCTCTGGATCAAAAAGGA BB
BBBbb_bb_abbbabbb`bbbbbaabbbbb`bbbbbbbabbabbbbb XT:A:R NM:i:1 SM:i:0 AM:i:0 X0:i:3 X1:i:0 XM:i:1 XO:i:0 XG:i:0 MD:Z:0T48 XA:Z:hg19_refGene_NM_020800,-1801,49M,1;hg
19_refGene_NM_001190241,-2021,49M,1;

[lh3]

If you map to human, 99.999% are mapped. Please read the manual page for the other question.

[edge]

Hi Yuan,

Do you mind to explain what is the exact meaning of 0, 4, 16 if I map my single-end read against reference transcript by using bowtie?
As I know, 4 is refer to unmapped.
How about 0 and 16 represent?
Thanks.

[win804]

0 means map in forward strand.
16 means map in reverse strand.

[dpryan]

Hi Yuan,

Do you mind to explain what is the exact meaning of 0, 4, 16 if I map my single-end read against reference transcript by using bowtie?
As I know, 4 is refer to unmapped.
How about 0 and 16 represent?
Thanks.

See the SAM specification. In short 16 means the read was reverse complemented and 0 means none of the flags were set, so the read mapped to the forward strand.

[edge]

Hi win804,

Many thanks for your advice.
I understand it now.
Thanks again.

[edge]

Hi dpryan,

Many thanks for your reply.
I understand it right now

[edge]

Hi win804,

Do you know what is the meaning of 20?
I run bwa to map my single-end read back to contig.
It shown 0, 4, 16, 20 in the sorted.bam T_T

Thanks for advice.

[edge]

Hi dpryan,

Do you know what is the meaning of 20?
I run bwa to map my single-end read back to contig.
It shown 0, 4, 16, 20 in the sorted.bam T_T

Thanks for advice.

[win804]

Hi Edge,
This page is probably useful for you: http://picard.sourceforge.net/explain-flags.html

20 means the read mapped in reverse strand and also the read is unmapped.
I know it's confusing. But the read is considered unmapped because it mapped to two different contigs (i.e. end of one contig and the start of another contig). You can check the read with the flag 20. It should be few bases from the end of the contig.

Thanks.