Hello,
i would like to only get reads that aligned to the forward / reverse strand separately. I am using this for now:
samtools view -F 20 ... : forward strand
samtools view -f 0x10 ... : reverse strand
Is this correct? With -F 20 i also ignore the not aligned reads.
Tnx!
Gregor
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Gregor Rot
Hey there,
I want to subtract the reads from forward and reverse strand, from a BAM file of Histone ChiP-seq data, to get the net reads mapping on either forward/reverse strand, how to do that. Any suggestions will be appreciated.
0x20 is if your data is paired end and will filter based on the strand of the mate. You want 0x10 for the query. For standard NGS data, mostly your pairs should be on the opposite strands anyway - except for weird events like DNA translocations, e.g., inversions.
[There is a slide (Using NGS Data to find chromosomal rearrangements) in this [PDF][1] to explain how the pairs will behave for these events.]
so do this for forward:
samtools view -F 0x10
and this for reverse:
samtools view -f 0x10
Use the -X flag in "samtools view" to convert the flag column from a numeric bit field to letters (as described here) like this:
% samtools view -Xf 0x10 accepted_hits.bam | head -1
24_727_529_F3 r chr1 2 0 50M * 0 0 ATCCAGCATCCAGCATCCAGCATCCAGCATCCAGCATCCAGCATCCAGCA @@!!",>4CS/!!;UPB!(QB>=EH5/!>T>9?S^ZUQZaZVY\_]^bcc NM:i:0 NH:i:31 CC:Z:= CP:i:9
% samtools view -XF 0x10 accepted_hits.bam | head -1
2118_596_811_F3 chr1 5367 3 50M * 0 0 CAGTGCAATCGTTTATAGACACAAACACCTTTTCCGCTGTTGATGGTGGT ___ZZHFZ\]PPRUb_ZNQUNOQYYP!!!#ROLPZSRNO@/GH2=NL;.. NM:i:0 NH:i:2 CC:Z:= CP:i:2868239
True - it was confusing as he mixed hex and dec values.
I found for using single end reads it could be written also as
samtools view -F 20 ... : forward strand
samtools view -f 16 ... : reverse strand
samtools view -f 4 ... : unmapped
And since I don't see it mentioned on this particular page, I'll add that tool at https://broadinstitute.github.io/picard/explain-flags.html is particularly helpful for making and deciphering these commands. The fact that the flag 20 includes both unmapped and those mapped to the reverse strand was not obvious to me at first. Bitwise is a compact but not immediately obvious way to indicate these sets. Slides 28-31 of Pierre Lindenbaums's presentation, which he highlighted in this post SAM flags meaning helped a lot.
Thanks! Specially, the URL from broadInstitute was very useful.
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Chirag Nepal
They forgot unmapped reads!
Please use these:
Forward
samtools view -c alignment.bam -h -F 20
Reverse
samtools view -c alignment.bam -h -f 16
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