Hello,
I am a bioinformatician working at the University of Pennsylvania and I just recently started the analysis of BiSulfite-seq data. I have with me a library of condition A paired-end sequenced in 2 different lanes and the same for condition B (i.e. 4 fastq files per condition).
I have used Bismark to align the reads and make the methylation calls. I then used BSmooth to smooth my data and call differentially methylated regions. After I do this and plot the smoothed data there are a few discrepancies which I am not sure what they mean.
1) I see straight lines (i.e. constant methylation %) in the smoothed plots of a lot of DMRs leading me to believe they are outliers of some kind. How do I know if these are real?
2) I see a lot of variability in the methylation % between the same library sequenced in different lanes. Is this a consequence of BSmooth/Bismark? Has anyone seen something similar happen?
3) Are there defined stringencies/cutoffs used for calling intergenic vs promoter DMRs at any point in the BSmooth pipeline?
Any help would be much appreciated!
I am a bioinformatician working at the University of Pennsylvania and I just recently started the analysis of BiSulfite-seq data. I have with me a library of condition A paired-end sequenced in 2 different lanes and the same for condition B (i.e. 4 fastq files per condition).
I have used Bismark to align the reads and make the methylation calls. I then used BSmooth to smooth my data and call differentially methylated regions. After I do this and plot the smoothed data there are a few discrepancies which I am not sure what they mean.
1) I see straight lines (i.e. constant methylation %) in the smoothed plots of a lot of DMRs leading me to believe they are outliers of some kind. How do I know if these are real?
2) I see a lot of variability in the methylation % between the same library sequenced in different lanes. Is this a consequence of BSmooth/Bismark? Has anyone seen something similar happen?
3) Are there defined stringencies/cutoffs used for calling intergenic vs promoter DMRs at any point in the BSmooth pipeline?
Any help would be much appreciated!
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