I am attempting to normalize my ChIPseq data with offsets and loess normalization as they appear to display trended bias and I want to visualise the effects of the normalization with the MA plot. I have generated the consensus peaks and the count object, which I then applied the dba.contrast() function, and subsequently dba.analyze(). I basically followed the steps on page 45 of the Diffbind documentation. https://bioconductor.org/packages/release/bioc/vignettes/DiffBind/inst/doc/DiffBind.pdf

However, I keep getting errors on "Blacklist error: Error: Invalid normalization" and "Unable to apply Blacklist/Greylist." despite removing the unwanted blacklists before hand. I also tried not removing blacklists beforehand and the code still didnt work.

Does anyone have any idea what could be causing the error and how I can fix it?

And if there is any recommendations or scripts of runnning successful offset and loess normalization?

Thank you!

library(DiffBind)
library(edgeR)
library(tidyverse)
library(dplyr)

samples <- read.csv('Sample.csv')

# create a dba object which contains the peaksets of the samples
dbObj <-dba(sampleSheet = samples)
dbObj

# ========= Obtain consensus peaks ===========
# Get the peakset for tumor and normal
tumor_normal_peaks <- dba.peakset(dbObj, 
                                  consensus=DBA_FACTOR, 
                                  minOverlap = 2) 

tumor_normal_overlap <- dba(tumor_normal_peaks,
                            mask=tumor_normal_peaks$masks$Consensus,
                            minOverlap = 2)

consensus_peaks <- dba.peakset(tumor_normal_overlap, 
                               bRetrieve = TRUE) 

consensus_peaks

# ============ Affinity binding analysis ================
# Extract the tumor-normal overlap peaks from the peaksets of each individual sample

tumor_normal_count_loess <- dba.count(dbObj, 
                                      peaks=consensus_peaks, #specify the peak source. Peaks retrieved from tumor_normal_overlap
                                      bUseSummarizeOverlaps = TRUE)

# Remove unwanted blacklists
tumor_normal_count_loess_blacklisted <-dba.blacklist(tumor_normal_count_loess, blacklist = DBA_BLACKLIST_HG38, greylist = FALSE)

# ============ Loess fit Normalisation ==================

tumor_normal_count_loess_blacklisted$config$AnalysisMethod <- DBA_EDGER
tumor_normal_count_loess_blacklisted$config$AnalysisMethod

tumor_normal_normalized_loess_blacklisted <- dba.normalize(tumor_normal_count_loess_blacklisted,
                                               offsets = TRUE)

norm <- dba.normalize(tumor_normal_normalized_loess_blacklisted, 
                      bRetrieve  = TRUE)
norm 

# ============ Modeling and contrast ================

tumor_normal_contrasted_loess_blacklisted <- dba.contrast(tumor_normal_normalized_loess_blacklisted,
                                                          categories=DBA_FACTOR, 
                                                          design = "~ Factor", 
                                                          contrast = c("Factor", "Tumor", "Normal"),  
                                                          minMembers = 2) 

tumor_normal_contrasted_loess_blacklisted

# ============ Differential analysis =================

tumor_normal_analyzed_loess_blacklisted <- dba.analyze(tumor_normal_contrasted_loess_blacklisted)

output

> tumor_normal_analyzed_loess <- dba.analyze(tumor_normal_contrasted_loess_blacklisted)
Applying Blacklist/Greylists...
Genome detected: Hsapiens.UCSC.hg38
Counting control reads for greylist...
rsession-arm64(44993) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(44994) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(44995) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(44996) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(44997) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(44998) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45008) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45007) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45011) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45009) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45012) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45010) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45137) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP152_1_INP.bam
rsession-arm64(45531) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45532) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45533) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45534) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45535) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45536) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45537) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45538) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 5124608 bp (0.17%)
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP152_3_INP.bam
rsession-arm64(45569) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45570) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45571) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45572) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45573) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45574) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45575) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45576) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 5351936 bp (0.17%)
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP152_4_INP.bam
rsession-arm64(45582) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45583) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45584) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45585) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45586) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45587) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45588) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45589) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 5366784 bp (0.17%)
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP262T2B_IN.bam
rsession-arm64(45595) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45596) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45597) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45598) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45599) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45600) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45601) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45602) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 4605439 bp (0.15%)
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP268N_INP.bam
rsession-arm64(45609) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45610) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45611) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45612) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45613) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45614) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45615) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45616) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 5565951 bp (0.18%)
Building greylist: /Volumes/T7ShieldSSD/hcc_h3k4me3/data/bam_k4/HEP276N_INP.bam
rsession-arm64(45621) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45622) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45623) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45624) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45625) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45626) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45627) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
rsession-arm64(45628) MallocStackLogging: can't turn off malloc stack logging because it was not enabled.
coverage: 4783616 bp (0.15%)
Control1: 428 ranges, 5124608 bases
Control2: 380 ranges, 5351936 bases
Control3: 376 ranges, 5366784 bases
Control4: 439 ranges, 4605439 bases
Control5: 387 ranges, 5565951 bases
Control6: 390 ranges, 4783616 bases
Master greylist: 780 ranges, 8692223 bases
Removed: 78 of 11422 intervals.
Re-normalizing...
Blacklist error: Error: Invalid normalization

Unable to apply Blacklist/Greylist.

It would be good to know what versions of the packages you are running (output of sessionInfo()), as this looks similar to an issue in somewhat earlier versions.

The issues appears to be with the greylist, not the blacklist. You can try either of two things:

1. Set greylist=TRUE in the call to dba.greylist() so that the greylist is computed prior to normalization. This was the bug that should be corrected in more recent versions.
2. Alternatively, set bGreylist=FALSE in the call to dba.analyze() to skip the greylisting step completely. (You can also set tumor_normal_analyzed_loess_blacklisted $config$doGreylist=FALSE to avoid having to re-specify this every time).

If this doesn't work, you may want to try turning off the blacklist as well (bBlacklist=FALSE).