DESeq2 normalization when using with derfinder
1
0
Entering edit mode
Debayan ▴ 20
@debayan-24308
Last seen 12 months ago
Spain

Hi, I have a question about whether I should set the sizeFactors to 1 prior to using DESeq2 on the counts obtained from derfinder. I have normalized bigwig files ( using deepTools ), which I use as input to derfinder. Following the instructions in the users guide : http://lcolladotor.github.io/derfinder/articles/derfinder-users-guide.html#expressed-regions-level-1 My doubt is that if the counts obtained by derfinder are already from normalized bigwig files, whether I should set the sizeFactors to 1 before running the DESeq function.

Thank you, Debayan

Code should be placed in three backticks as shown below


# include your problematic code here with any corresponding output 
# please also include the results of running the following in an R session 

## Determine the files to use and fix the names
files_RZ <- list.files(path="C:/Users/rnaseq_RZ/", pattern=".*bw$",full.names=T)

names(files_RZ) <- gsub(".deepTools.bw", "", basename(files_RZ))

## Load the data from disk -- On Windows you have to load data from Bam files
chrs=paste("chr",c(1:22,"X","Y","M"),sep="")
fullCov_RZ <- fullCoverage(files = files_RZ, chrs=chrs, verbose = FALSE)

## Get the region matrix of Expressed Regions (ERs)
regionMat_RZ <- regionMatrix(fullCov_RZ, cutoff = 30, L = 100, verbose = FALSE)

# Combine into a single one

## First extract the data
regs <- unlist(GRangesList(lapply(regionMat_RZ, '[[', 'regions')))
covMat <- do.call(rbind, lapply(regionMat_RZ, '[[', 'coverageMatrix'))
## Force the names to match
names(regs) <- rownames(covMat) <- seq_len(length(regs))
## Combine into a list (not really needed)
mergedRegionMat_RZ <- list('regions' = regs, 'coverageMatrix' = covMat)

## Round matrix
counts_RZ <- round(mergedRegionMat_RZ$coverageMatrix)

##Design
coldata=data.frame(factor(c("1","2","2","2","1","1","2","1","3","3","3","3")))

colnames(coldata)="condition"
rownames(coldata)=colnames(counts_RZ)

dds_RZ <- DESeqDataSetFromMatrix(countData = counts_RZ,
                              colData = coldata,
                              design = ~ condition)

dds2_RZ <- DESeq(dds_RZ)


sessionInfo( )
R version 4.0.3 (2020-10-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 19042)

Matrix products: default

locale:
[1] LC_COLLATE=English_United States.1252  LC_CTYPE=English_United States.1252   
[3] LC_MONETARY=English_United States.1252 LC_NUMERIC=C                          
[5] LC_TIME=English_United States.1252    

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

other attached packages:
 [1] reshape2_1.4.4              forcats_0.5.0               purrr_0.3.4                
 [4] readr_1.4.0                 tidyr_1.1.2                 tidyverse_1.3.0            
 [7] derfinder_1.24.2            ggrepel_0.8.2               DESeq2_1.30.0              
[10] SummarizedExperiment_1.20.0 Biobase_2.50.0              MatrixGenerics_1.2.0       
[13] matrixStats_0.57.0          GenomicRanges_1.42.0        GenomeInfoDb_1.26.2        
[16] IRanges_2.24.0              S4Vectors_0.28.1            BiocGenerics_0.36.0        
[19] leaflet_2.0.4.1             stringr_1.4.0               dplyr_1.0.2                
[22] ggplot2_3.3.3               tibble_3.0.4                RColorBrewer_1.1-2         
[25] shiny_1.6.0                

loaded via a namespace (and not attached):
  [1] readxl_1.3.1             backports_1.2.0          Hmisc_4.5-0              BiocFileCache_1.14.0    
  [5] plyr_1.8.6               lazyeval_0.2.2           sp_1.4-5                 splines_4.0.3           
  [9] BiocParallel_1.24.1      crosstalk_1.1.1          digest_0.6.27            foreach_1.5.1           
 [13] ensembldb_2.14.1         htmltools_0.5.1.1        viridis_0.5.1            leaflet.providers_1.9.0 
 [17] magrittr_2.0.1           checkmate_2.0.0          memoise_2.0.0            BSgenome_1.58.0         
 [21] cluster_2.1.0            Biostrings_2.58.0        annotate_1.68.0          modelr_0.1.8            
 [25] askpass_1.1              prettyunits_1.1.1        jpeg_0.1-8.1             colorspace_2.0-0        
 [29] rvest_0.3.6              blob_1.2.1               rappdirs_0.3.1           haven_2.3.1             
 [33] xfun_0.25                crayon_1.3.4             RCurl_1.98-1.2           jsonlite_1.7.1          
 [37] genefilter_1.72.0        survival_3.2-7           VariantAnnotation_1.36.0 iterators_1.0.13        
 [41] glue_1.4.2               gtable_0.3.0             zlibbioc_1.36.0          XVector_0.30.0          
 [45] DelayedArray_0.16.0      scales_1.1.1             DBI_1.1.0                rngtools_1.5            
 [49] derfinderHelper_1.24.1   Rcpp_1.0.5               viridisLite_0.3.0        xtable_1.8-4            
 [53] progress_1.2.2           htmlTable_2.2.1          bumphunter_1.32.0        foreign_0.8-80          
 [57] bit_4.0.4                Formula_1.2-4            htmlwidgets_1.5.3        httr_1.4.2              
 [61] ellipsis_0.3.1           pkgconfig_2.0.3          XML_3.99-0.5             farver_2.0.3            
 [65] nnet_7.3-14              sass_0.4.0               dbplyr_2.0.0             locfit_1.5-9.4          
 [69] tidyselect_1.1.0         labeling_0.4.2           rlang_0.4.11             later_1.2.0             
 [73] AnnotationDbi_1.52.0     cellranger_1.1.0         munsell_0.5.0            tools_4.0.3             
 [77] cachem_1.0.5             cli_3.0.1                generics_0.1.0           RSQLite_2.2.1           
 [81] broom_0.7.6              fastmap_1.1.0            yaml_2.2.1               fs_1.5.0                
 [85] knitr_1.33               bit64_4.0.5              AnnotationFilter_1.14.0  doRNG_1.8.2             
 [89] mime_0.9                 xml2_1.3.2               biomaRt_2.46.0           compiler_4.0.3          
 [93] rstudioapi_0.13          curl_4.3                 png_0.1-7                reprex_0.3.0            
 [97] geneplotter_1.68.0       bslib_0.2.5.1            stringi_1.5.3            GenomicFeatures_1.42.1  
[101] GenomicFiles_1.26.0      lattice_0.20-41          ProtGenerics_1.22.0      Matrix_1.2-18           
[105] vctrs_0.3.5              pillar_1.4.7             lifecycle_1.0.0          jquerylib_0.1.4         
[109] data.table_1.13.4        bitops_1.0-6             raster_3.4-13            qvalue_2.22.0           
[113] httpuv_1.6.1             rtracklayer_1.49.5       R6_2.5.0                 latticeExtra_0.6-29     
[117] promises_1.2.0.1         gridExtra_2.3            codetools_0.2-16         assertthat_0.2.1        
[121] openssl_1.4.3            withr_2.4.2              GenomicAlignments_1.26.0 Rsamtools_2.6.0         
[125] GenomeInfoDbData_1.2.4   hms_0.5.3                grid_4.0.3               rpart_4.1-15            
[129] lubridate_1.7.9.2        base64enc_0.1-3          tinytex_0.33
Normalization DESeq2 derfinder • 942 views
ADD COMMENT
3
Entering edit mode
@mikelove
Last seen 22 hours ago
United States

In the derfinder vignette they do not set size factors to 1, but use our size factor estimation.

ADD COMMENT
0
Entering edit mode

Thanks a lot for the clarification.

ADD REPLY

Login before adding your answer.

Traffic: 721 users visited in the last hour
Help About
FAQ
Access RSS
API
Stats

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.

Powered by the version 2.3.6