I have few doubts using MACS2
1. macs2 callpeak -t chip.bedgraph -c input.bedgraph --outdir Input_test -B --nomodel --SPMR
This step generates a control lambda.bdg file and a treated pileup bdg file.
As per my understanding --SPMR normalizes the dataset to 1M reads . So , if it is normalizes, can we
convert this data to bigwig file and then visualize the control and treated sample? Does it make sense?
2. macs2 bdgcmp -t treated_pileup.bdg -c control_lambda.bdg -m logLR --outdir Ba-HW-bdgcmp -o out.bdg
This command is used to remove background noise between the control and treated set. If this is the case what step (1) does? Is it not normalized in step1? If so what the use of --SPMR.
More to my confusion bdgcmp has --scaling factor as an option. To my understanding this is also used for normalization. Again if I want to use logLR for bdgcmp i have to provide -p parameter., but it says this parameter is applied after normalization of sequencing depth. How should i do? I am unable to understand. Please guide me.
1. Which step does normalization, --SPMR or scaling factor. If scaling factor, how to estimate the value?
2. Which files should i take for comparing the chipped and input data. callpeak generates .bdg for control and treated and after that bdgcmp generates one more .bdg file. which files should be considered for visualization using IGV.
3. If I want to provide -m logLR during bgdcmp , I have to provide -p also. Inorder to use -p, the data has to be normalized to sequencing depth?
I am sorry, I am quite new to macs2 so have lot of confusion.
Your inputs will be highly appreciated.