Question: MACS2 reads report inconsistent with samtools flagstat
0
gravatar for Ming Tang
4.0 years ago by
Ming Tang2.5k
Houston/MD Anderson Cancer Center
Ming Tang2.5k wrote:

Hi there,

I have read [here](https://groups.google.com/forum/#!searchin/macs-announcement/duplicated$20reads/macs-announcement/7E8_Hg_irCQ/-wjSg7H55QIJ) and [here](https://groups.google.com/forum/#!searchin/macs-announcement/samtools$20flagstat/macs-announcement/Vy03--fXBSY/0rPbL7-HCocJ

But, I am still confused. 

My IP bam flagstat:

18880801 + 0 in total (QC-passed reads + QC-failed reads)

253218 + 0 duplicates

14130154 + 0 mapped (74.84%:-nan%)

0 + 0 paired in sequencing

0 + 0 read1

0 + 0 read2

0 + 0 properly paired (-nan%:-nan%)

0 + 0 with itself and mate mapped

0 + 0 singletons (-nan%:-nan%)

0 + 0 with mate mapped to a different chr

0 + 0 with mate mapped to a different chr (mapQ>=5)

MACS2 report:

# tag size is determined as 36 bps

# total tags in treatment: 8308438

# tags after filtering in treatment: 8005485

# maximum duplicate tags at the same position in treatment = 1

# Redundant rate in treatment: 0.04

So, after remove duplicates:

14130154 (mapped) - 253218(duplicates) = 13876936.

This number is still much bigger than 8308438. 

So, why is that? and why MACS2 further filter down to 8005485?

 

Thanks,

Ming

chip-seq macs2 • 1.6k views
ADD COMMENTlink modified 4.0 years ago • written 4.0 years ago by Ming Tang2.5k

It might be that macs2 removes reads with mapq below a certain threshold and other uncertain reads. Try to run "samtools view -c -F3844 -q 5 myaln.bam" and see if you get a number closer to macs'. -F3844 filters out:

read unmapped
not primary alignment
read fails platform/vendor quality checks
read is PCR or optical duplicate
supplementary alignment

ADD REPLYlink written 4.0 years ago by dariober10k

Thanks for your comment. but MACS2 does not check read quality: https://groups.google.com/forum/#!topic/macs-announcement/ASQ3GsXQC1c

ADD REPLYlink written 4.0 years ago by Ming Tang2.5k

I run that command, and it gives me 14,130,154 

still much bigger than the 8 million reads MACS2 retains.

ADD REPLYlink written 4.0 years ago by Ming Tang2.5k
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