Q: Is it necessary to use IgG as control for CUT&TAG data

[leafyeszjl]

Hi, my data is CUT&Tag-sequencing, but I don't know should I add IgG sample as control for peak calling when using macs2. In my experiment, L9 was the IgG sample, and L1\L2\L3\L4 sample were just different in cells input (100，1k,1w and 10 w respectively). Other experimental conditions are the same. The alignment results was as table 1. when I did not add IgG control, the peaks number was as table 2. The number of peaks increased with the amount of cells. However, it get the opposite result when using the L9 as IgG control (Table3). I don't know which result is correct, could you give me any advice? (1) Table1 :Alignment results

Sample	L1	L2	L3	L4	L9
Clean reads pair	23352501	14016997	8588937	12006095	7104387
Mapped reads pair	21165340	12074669	7180872	11589712	6476417
Mapping rate(%)	90.63	86.14	83.61	96.53	91.16
Duplicate reads	40234544	22294654	10839854	6596992	11982356
Duplicate rate(%)	95.05	92.32	75.48	28.46	92.51
chrMT reads	570956	401178	397724	431596	1452286
chrMT rate(%)	1.35	1.66	2.77	1.86	11.21

(2)Table2: Number of peaks calling by macs2 without IgG. macs2 callpeak -t L2.rmMT.bam -g hs -f BAMPE -n L2 -p 1e-05

Sample	Peaks_num
L1	2204
L2	2800
L3	6531
L4	18018

(3)Table3 :Number of peaks calling by macs2 with IgG. macs2 callpeak -t L2.rmMT.bam -c L9.rmMT.bam -g hs -f BAMPE -n L2 -p 1e-05

Sample	Peaks_number
L1	34841
L2	44386
L3	8201
L4	5011

(4) TSS heatmap

[jamesc99]

Hi, my data is CUT&Tag-sequencing, but I don't know should I add IgG sample as control for peak calling when using macs2. In my experiment, L9 was the IgG sample, and L1\L2\L3\L4 sample were just different in cells input (100，1k,1w and 10 w respectively). Other experimental conditions are the same. The alignment results was as table 1. when I did not add IgG control, the peaks number was as table 2. The number of peaks increased with the amount of cells. However, it get the opposite result when using the L9 as IgG control (Table3). I don't know which result is correct, could you give me any advice? (1) Table1 :Alignment results

Sample L1 L2 L3 L4 L9 Clean reads pair 23352501 14016997 8588937 12006095 7104387 Mapped reads pair 21165340 12074669 7180872 11589712 6476417 Mapping rate(%) 90.63 86.14 83.61 96.53 91.16 Duplicate reads 40234544 22294654 10839854 6596992 11982356 Duplicate rate(%) 95.05 92.32 75.48 28.46 92.51 chrMT reads 570956 401178 397724 431596 1452286 chrMT rate(%) 1.35 1.66 2.77 1.86 11.21 (2)Table2: Number of peaks calling by macs2 without IgG. macs2 callpeak -t L2.rmMT.bam -g hs -f BAMPE -n L2 -p 1e-05

Sample Peaks_num L1 2204 L2 2800 L3 6531 L4 18018 (3)Table3 :Number of peaks calling by macs2 with IgG. macs2 callpeak -t L2.rmMT.bam -c L9.rmMT.bam -g hs -f BAMPE -n L2 -p 1e-05

Sample Peaks_number L1 34841 L2 44386 L3 8201 L4 5011 (4) TSS heatmap

Hey, Have you ever solved this issue? I encountered same problem with you. But my situation is that the detected peaks number with IgG control are much lower than those without IgG control. Thanks!