Dear Biostar users.
This is my samtools flagstat output.
39219750 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
39219750 + 0 mapped (100.00% : N/A)
39219750 + 0 paired in sequencing
19658242 + 0 read1
19561508 + 0 read2
39219750 + 0 properly paired (100.00% : N/A)
39219750 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
1 + 0 with mate mapped to a different chr
1 + 0 with mate mapped to a different chr (mapQ>=5)
In this situation, why are not read1 and read2 read identical?
ADD) I want to get paired read and properly mapped read. so I run this command in linux
samtools view -b -f 3 -F 780 input.bam > properly_ex_qual_unmap_1_mapped.bam and flagstat result (above) is from this bam.
I can't understand this result. Because in flagstat result all reads were properly mapped and all reads were mapped.
Thanks for your help.
Simply because some reads did not mapped under the conditions indicated by the program
Thank you Franco.
If so, can I extract identical read1(forward) and read2(reverse) from this bam?
Thank you for your reply.
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Thanks for Franco, Yague, Ryan.
1st. According to Yague and Franco advice, I run samtools view and flagstat
1) samtools view -b -f 2 -F 12 input.bam > f2F12.bam
39219750 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
39219750 + 0 mapped (100.00% : N/A)
39219750 + 0 paired in sequencing
19658242 + 0 read1
19561508 + 0 read2
39219750 + 0 properly paired (100.00% : N/A)
39219750 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
1 + 0 with mate mapped to a different chr
1 + 0 with mate mapped to a different chr (mapQ>=5)
2) samtools view -b -F 12 input.bam > F12.bam
40294316 + 0 in total (QC-passed reads + QC-failed reads)
0 + 0 secondary
0 + 0 supplementary
0 + 0 duplicates
40294316 + 0 mapped (100.00% : N/A)
39486133 + 0 paired in sequencing
19793464 + 0 read1
19692669 + 0 read2
39219750 + 0 properly paired (99.33% : N/A)
39486133 + 0 with itself and mate mapped
0 + 0 singletons (0.00% : N/A)
219437 + 0 with mate mapped to a different chr
219437 + 0 with mate mapped to a different chr (mapQ>=5)
this result and my 1st question's output are same. The reason why my 1st question's output is from bam (samtools view -b -f 3 -F 780 input.bam > properly_ex_qual_unmap_1_mapped.bam ).
2nd. According to Ryan's advice, I checked my raw fastq files. But this bam file is too old, so raw fastq file was deleted.
3rd. In Ryan's post (link: https://www.biostars.org/p/233441/#233455), different number of # read1 and # read2 is related to singletons. But my flagstat result seems like "no singleton".
I need only paired reads ( identical number of read1 and read2 ). But I can't find method.
Your all answer is helped me. Thanks.
Hello.
I think I found a problem.
In f2F12.bam file ( this bam file was made of "samtools view -f 2 -F 12 input.bam > f2F12.bam") [samtools -f 2 -F 2 input.bam > f2F2.bam --> mistake],
a read's flag value is 99.
According to https://broadinstitute.github.io/picard/explain-flags.html, 99 mean follwing 4things.
But in f2F12.bam file, the read have no pair reads......
I think other reads are same reason.
Can I get only exactly and truly paired reads using program or command? delete unmatched read?
I think my bam file is too bad.
Thanks
Did you use
bwa mem? If so, the difference is due to supplemental alignments.Thank you Ryan.
I checked my bamfile Header. This bam file is maybe made of bwa aln and samse.
I can't understand this situation...
Because in the flagstat output, they are all properly paired and all mapped and all paired in sequencing . :(
Then, how can I extract exactly identical read1 and read2?
Thank you for your reply.
what do you mean by identical reads?
I'm sorry for my confused word.
I mean
I want to get only paired reads that have identical header name.
If so, the number of read1 and read2 will be identical.
Isn't it?
Thanks
Go to this page Meaning of sam flags Every mapped read containing the 2 value in their flag, is a read mapped in a proper pair. So by using
you theoretically can get your properly mapped reads
alternatively, to extract the mapped reads whose mates are also mapped, u can use
Check the number of lines in the two original fastq files you had. Perhaps one has a few extra reads.
Did you actually do
-f 2 -F 2or did you mean-f 2 -F 12?-f 2and-F 2are opposites, so it's unclear what the results of that would be.oh I'm sorry for my mistake.
I do "samtools view -f 2 -F 12".
You might need to just post the BAM file somewhere. Then we can have a look at what's going on. My guess is that this file was filtered at some point, which is why there's a discrepancy in the mates.
You can also try to use
samtools fixmateto update mate-related flags before runningflagstats.