Help!!! Different results by using extendReads or not

[icanwinwyz]

Dear deeptools developer, Thanks for developing such a great tool! I recently ran a Cut & Tag experiment(2 X 150bp) and want to use the plots from deeptools to show the signal distribution of different histone markers on TSS regions. However, I got different results when I used "extendReads" or not in bamCoverage function with BPM normalization. Please see the plot shown below:

The plot showed that the male sample (the "m" in the sample name) has a higher reads intensity than female sample (the "f" in the sample name) when I used extendReads, but the direction is opposite when I deactivated extendReads. I thought it was possibly caused by the quantification issue that the mate reads in properly paired reads were quantified twice. So I included "--samFlagInclude 66" in bamCoverage to quantify the mate reads only once with extendReads activated. However, the result showed similar as the one activating extendReads without samFlagInclude parameter (see below):

I am wondering why "extendReads" caused so much different results? This is important since it would results in different interpretation for biology.

deeptools version: 3.5.1 python version: 3.9.13

Here are the codes: ###with extendReads activated bamCoverage --bam .bam -o .extent.bw --ignoreDuplicates --normalizeUsing BPM --blackListFileName ../blacklist.merge.bed --binSize 10 --ignoreForNormalization chrX chrY chrM --extendReads

###with extendReads deactivated bamCoverage --bam .bam -o .extent.bw --ignoreDuplicates --normalizeUsing BPM --blackListFileName ../blacklist.merge.bed --binSize 10 --ignoreForNormalization chrX chrY chrM

###with samFlagInclude 66 bamCoverage --bam .bam -o .extent_samflag66.bw --ignoreDuplicates --normalizeUsing BPM --blackListFileName ../blacklist.merge.bed --binSize 10 --ignoreForNormalization chrX chrY chrM

###computeMatrix codes computeMatrix reference-point --referencePoint TSS -p 20 -b 2500 -a 2500 -R .bed -S .bw --missingDataAsZero -o

.gz

I look forward to your reply! Many thanks in advance!!

Joe Wang