I thought it might be worthwhile to check how well the DiffBind analysis using DESeq2 fits the assumptions needed. The data comes from ChIP-Seq. I performed a DESeq2 analysis, using DiffBind fetched a DESeq2 object from its results, and drew the dispersion plot.

The fitted function has a "hump" meaning that the dispersion becomes higher with the mean for some portion of the graph.

I think that in an RNA-Seq one should be worried if such a graph would be produced. How worrisome is it in ChIP-Seq, in diffbind?

Also, since I cannot assume balanced changes in binding between the various conditions, I have normalized by the total library size - not by DESeq2 native normalization. So unsurprisingly, the distribution is different for the different samples. I think there is no other option - but are these invalidated assumptions that ruin the analysis?

dbObj_b_contrast_qc <- dba.contrast(dbObj_b_norm,design="~Condition")
dbObj_b_res_qc <- dba.analyze(dbObj_b_contrast_qc)
dds_qc <- dba.analyze(dbObj_b_res_qc, bRetrieveAnalysis = T)
plotDispEsts(dds_qc)
normc <- as.data.frame(counts(dds_qc, normalized=T))
myBoxplot(normc) # a function plotting boxplots with ggplot2

print(dds_qc$Condition)
sessionInfo()

Output:

[1] WT         WT         WT         Mut        Mut        Mut       
[7] Rescue     Rescue     Rescue_Mut Rescue_Mut Rescue_Mut
Levels: WT Mut Rescue Rescue_Mut



R version 4.0.4 (2021-02-15)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 20.04.2 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/blas/libblas.so.3.9.0
LAPACK: /usr/lib/x86_64-linux-gnu/lapack/liblapack.so.3.9.0

locale:
 [1] LC_CTYPE=en_IL.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=en_IL.UTF-8        LC_COLLATE=en_IL.UTF-8    
 [5] LC_MONETARY=en_IL.UTF-8    LC_MESSAGES=en_IL.UTF-8   
 [7] LC_PAPER=en_IL.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=en_IL.UTF-8 LC_IDENTIFICATION=C       

attached base packages:
 [1] parallel  stats4    grid      stats     graphics  grDevices utils    
 [8] datasets  methods   base     

other attached packages:
 [1] limma_3.46.0                rgl_0.105.13               
 [3] DiffBind_3.0.13             DESeq2_1.30.1              
 [5] SummarizedExperiment_1.20.0 Biobase_2.50.0             
 [7] MatrixGenerics_1.2.1        matrixStats_0.58.0         
 [9] GenomicRanges_1.42.0        GenomeInfoDb_1.26.2        
[11] IRanges_2.24.1              S4Vectors_0.28.1           
[13] BiocGenerics_0.36.0         RColorBrewer_1.1-2         
[15] pheatmap_1.0.12             ggrepel_0.9.1              
[17] ggplot2_3.3.3              

loaded via a namespace (and not attached):
  [1] backports_1.2.1          GOstats_2.56.0          
  [3] BiocFileCache_1.14.0     plyr_1.8.6              
  [5] GSEABase_1.52.1          splines_4.0.4           
  [7] crosstalk_1.1.1          BiocParallel_1.24.1     
  [9] amap_0.8-18              digest_0.6.27           
 [11] htmltools_0.5.1.1        invgamma_1.1            
 [13] GO.db_3.12.1             SQUAREM_2021.1          
 [15] fansi_0.4.2              magrittr_2.0.1          
 [17] checkmate_2.0.0          memoise_2.0.0           
 [19] BSgenome_1.58.0          base64url_1.4           
 [21] Biostrings_2.58.0        annotate_1.68.0         
 [23] systemPipeR_1.24.3       askpass_1.1             
 [25] bdsmatrix_1.3-4          prettyunits_1.1.1       
 [27] jpeg_0.1-8.1             colorspace_2.0-0        
 [29] blob_1.2.1               rappdirs_0.3.3          
 [31] apeglm_1.12.0            xfun_0.21               
 [33] dplyr_1.0.4              crayon_1.4.1            
 [35] RCurl_1.98-1.2           jsonlite_1.7.2          
 [37] graph_1.68.0             genefilter_1.72.1       
 [39] brew_1.0-6               survival_3.2-7          
 [41] VariantAnnotation_1.36.0 glue_1.4.2              
 [43] gtable_0.3.0             zlibbioc_1.36.0         
 [45] XVector_0.30.0           webshot_0.5.2           
 [47] DelayedArray_0.16.1      V8_3.4.0                
 [49] Rgraphviz_2.34.0         scales_1.1.1            
 [51] mvtnorm_1.1-1            DBI_1.1.1               
 [53] edgeR_3.32.1             miniUI_0.1.1.1          
 [55] Rcpp_1.0.6               xtable_1.8-4            
 [57] progress_1.2.2           emdbook_1.3.12          
 [59] bit_4.0.4                rsvg_2.1                
 [61] AnnotationForge_1.32.0   truncnorm_1.0-8         
 [63] htmlwidgets_1.5.3        httr_1.4.2              
 [65] gplots_3.1.1             ellipsis_0.3.1          
 [67] farver_2.0.3             pkgconfig_2.0.3         
 [69] XML_3.99-0.5             sass_0.3.1              
 [71] dbplyr_2.1.0             locfit_1.5-9.4          
 [73] utf8_1.1.4               labeling_0.4.2          
 [75] manipulateWidget_0.10.1  later_1.1.0.1           
 [77] tidyselect_1.1.0         rlang_0.4.10            
 [79] AnnotationDbi_1.52.0     munsell_0.5.0           
 [81] tools_4.0.4              cachem_1.0.4            
 [83] cli_2.3.0                generics_0.1.0          
 [85] RSQLite_2.2.3            evaluate_0.14           
 [87] stringr_1.4.0            fastmap_1.1.0           
 [89] yaml_2.2.1               knitr_1.31              
 [91] bit64_4.0.5              caTools_1.18.1          
 [93] purrr_0.3.4              RBGL_1.66.0             
 [95] mime_0.10                xml2_1.3.2              
 [97] biomaRt_2.46.3           compiler_4.0.4          
 [99] rstudioapi_0.13          curl_4.3                
[101] png_0.1-7                geneplotter_1.68.0      
[103] tibble_3.0.6             bslib_0.2.4             
[105] stringi_1.5.3            GenomicFeatures_1.42.1  
[107] lattice_0.20-41          Matrix_1.3-2            
[109] vctrs_0.3.6              pillar_1.5.0            
[111] lifecycle_1.0.0          jquerylib_0.1.3         
[113] irlba_2.3.3              data.table_1.14.0       
[115] bitops_1.0-6             httpuv_1.5.5            
[117] rtracklayer_1.50.0       R6_2.5.0                
[119] latticeExtra_0.6-29      hwriter_1.3.2           
[121] promises_1.2.0.1         ShortRead_1.48.0        
[123] KernSmooth_2.23-18       MASS_7.3-53.1           
[125] gtools_3.8.2             assertthat_0.2.1        
[127] openssl_1.4.3            Category_2.56.0         
[129] rjson_0.2.20             withr_2.4.1             
[131] GenomicAlignments_1.26.0 batchtools_0.9.15       
[133] Rsamtools_2.6.0          GenomeInfoDbData_1.2.4  
[135] hms_1.0.0                DOT_0.1                 
[137] coda_0.19-4              rmarkdown_2.7           
[139] GreyListChIP_1.22.0      ashr_2.2-47             
[141] mixsqp_0.3-43            bbmle_1.0.23.1          
[143] shiny_1.6.0              numDeriv_2016.8-1.1     
[145] tinytex_0.29

Just my two cents (not being DiffBind/DESeq2 developer): If your libraries are expected to be very different it is especially important to NOT use just plain per-million but something that attempts to correct for composition. I would inspect pairwise MA-plots between samples to see how the data look. Usually MAs have an arrowhead-like shape and the point on the very far are the regions with very large counts which often do not (or only very modestly) change, therefore they (so the "head" of the arrowhead) should be somewhat centered along y=0. You can then explore normalization techniques followed by MA-plots again and visual inspection which one centers these regions best at y=0. It might be a good idea to subset the data to only like the 25% regions with the highest counts (again = the regions farright on the MA) to calculate the size factors, and then feed them manually into your DESeq2 object. Not sure how this goes in DiffBind. I found this useful for some of my datasets (using DESeq2 directly) where composition was kind of impalanced between up- and downregulated regions, and using the full dataset for normalization did not properly center the aforementioned regions at y=0. As you usually have more points on the left than right of the MA-plots (in my understanding) these point have kind of more "weight" to influence normalization, and if these regions change a lot between samples then it might be necessary to perform the aforementioned subsetting to high-count regions assuming that these do not change / are robust estimators of non-changing regions. tl;dr, hope this makes sense to you.