DESeq2: All samples have 0 counts for all genes. check the counting script
Entering edit mode
Sonia • 0
Last seen 2.7 years ago
United Kingdom

I am having problems importing my HTSeq count data- it tells me the counts are zero when this is clearly not the case when head outputs:

    GeneID WTCHG_862660_71955267
1     A1BG                   148
2 A1BG-AS1                   196
3     A1CF                     0
4      A2M                     1
5  A2M-AS1                    79
6    A2ML1                     0

This is the script:

#this works
metadata <- read.csv("Meta_data_clones_vs_vbneg.csv")

#this adds in a column to say what the file name is in the metadata file
metadata = mutate(metadata, countFile = paste0(metadata$SampleName, ".stranded-counts.txt"))
metadata <- lapply(metadata, as.character)

#this works and makes the sample table
mysampleTable <- data.frame(sampleName = metadata$SampleName, fileName = metadata$countFile, condition = metadata$Condition)

#this creates the table of counts
DESeq2Table <- DESeqDataSetFromHTSeqCount(sampleTable = mysampleTable, directory = "H:/R/htseq_counts_vbneg_pos/", design = ~condition, ignoreRank = FALSE)

# include your problematic code here with any corresponding output 
#Error in DESeqDataSet(se, design = design, ignoreRank) : 
  all samples have 0 counts for all genes. check the counting script.

# please also include the results of running the following in an R session 

sessionInfo( )

R version 4.1.1 (2021-08-10)
Platform: x86_64-w64-mingw32/x64 (64-bit)
Running under: Windows 10 x64 (build 18363)

Matrix products: default

[1] LC_COLLATE=English_United Kingdom.1252 
[2] LC_CTYPE=English_United Kingdom.1252   
[3] LC_MONETARY=English_United Kingdom.1252
[4] LC_NUMERIC=C                           
[5] LC_TIME=English_United Kingdom.1252    

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

other attached packages:
 [1] DESeq2_1.32.0               SummarizedExperiment_1.22.0
 [3] Biobase_2.52.0              MatrixGenerics_1.4.3       
 [5] matrixStats_0.61.0          GenomicRanges_1.44.0       
 [7] GenomeInfoDb_1.28.4         forcats_0.5.1              
 [9] stringr_1.4.0               dplyr_1.0.7                
[11] purrr_0.3.4                 readr_2.0.1                
[13] tidyr_1.1.3                 tibble_3.1.4               
[15] ggplot2_3.3.5               tidyverse_1.3.1            
[17] IRanges_2.26.0              S4Vectors_0.30.0           
[19] BiocGenerics_0.38.0        

loaded via a namespace (and not attached):
 [1] bitops_1.0-7           fs_1.5.0               usethis_2.0.1         
 [4] devtools_2.4.2         bit64_4.0.5            lubridate_1.7.10      
 [7] RColorBrewer_1.1-2     httr_1.4.2             rprojroot_2.0.2       
[10] tools_4.1.1            backports_1.2.1        utf8_1.2.2            
[13] R6_2.5.1               DBI_1.1.1              colorspace_2.0-2      
[16] withr_2.4.2            tidyselect_1.1.1       prettyunits_1.1.1     
[19] processx_3.5.2         bit_4.0.4              compiler_4.1.1        
[22] cli_3.0.1              rvest_1.0.1            xml2_1.3.2            
[25] desc_1.4.0             DelayedArray_0.18.0    scales_1.1.1          
[28] genefilter_1.74.0      callr_3.7.0            XVector_0.32.0        
[31] pkgconfig_2.0.3        sessioninfo_1.1.1      dbplyr_2.1.1          
[34] fastmap_1.1.0          rlang_0.4.11           readxl_1.3.1          
[37] rstudioapi_0.13        RSQLite_2.2.8          generics_0.1.0        
[40] jsonlite_1.7.2         BiocParallel_1.26.2    RCurl_1.98-1.5        
[43] magrittr_2.0.1         GenomeInfoDbData_1.2.6 Matrix_1.3-4          
[46] Rcpp_1.0.7             munsell_0.5.0          fansi_0.5.0           
[49] lifecycle_1.0.1        stringi_1.7.4          zlibbioc_1.38.0       
[52] pkgbuild_1.2.0         blob_1.2.2             grid_4.1.1            
[55] crayon_1.4.1           lattice_0.20-45        splines_4.1.1         
[58] Biostrings_2.60.2      haven_2.4.3            annotate_1.70.0       
[61] KEGGREST_1.32.0        hms_1.1.1              locfit_1.5-9.4        
[64] ps_1.6.0               pillar_1.6.2           pkgload_1.2.2         
[67] geneplotter_1.70.0     reprex_2.0.1           XML_3.99-0.8          
[70] glue_1.4.2             remotes_2.4.1          BiocManager_1.30.16   
[73] modelr_0.1.8           png_0.1-7              vctrs_0.3.8           
[76] tzdb_0.1.2             testthat_3.0.4         cellranger_1.1.0      
[79] gtable_0.3.0           assertthat_0.2.1       cachem_1.0.6          
[82] xtable_1.8-4           broom_0.7.9            survival_3.2-13       
[85] AnnotationDbi_1.54.1   memoise_2.0.0          ellipsis_0.3.2

I tried manually adding a column with row numbers (using the rowid_to_column) function but it returned the same error message.

Anyone help?? I've going round and round for days with this.

DESeq2 RStudio • 2.4k views
Entering edit mode

I think it would be hard to help you as it stands, could you share what mysampleTable looks like ? Have you checked the filenames correctly match with your directory ?

Entering edit mode

This is the head of mysampleTable and yes the file names do match.

      sampleName                                  fileName condition
1 WTCHG_862660_71955267 WTCHG_862660_71955267.stranded-counts.txt     Vbneg
2 WTCHG_862660_71965268 WTCHG_862660_71965268.stranded-counts.txt     Vbpos
3 WTCHG_862660_71985270 WTCHG_862660_71985270.stranded-counts.txt     Vbneg
4 WTCHG_862660_71995271 WTCHG_862660_71995271.stranded-counts.txt     Vbpos
5 WTCHG_862660_72035275 WTCHG_862660_72035275.stranded-counts.txt     Vbneg
6 WTCHG_862660_72045276 WTCHG_862660_72045276.stranded-counts.txt     Vbpos
Entering edit mode

Everything seems fine here, so I suppose there might be a problem with the txt files. Is there any row header in your txt files ? If yes, just remove it and it might solve the issue

Entering edit mode

Yes! There was a header in the text files that just read '0'. I removed it manually and it works fine now.

Entering edit mode
swbarnes2 ★ 1.4k
Last seen 2 days ago
San Diego

If you look at the examples in the vignette, the rownames of the sample table and counts files are not just row numbers.


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