DESeq2: All samples have 0 counts for all genes. check the counting script
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Sonia • 0
@2f3f6904
Last seen 3.0 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:

>head(WTCHG_862660_71955267)
    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")
metadata
print(metadata)

#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)
metadata

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

#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

locale:
[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 • 2.7k views
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0
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 ?

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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
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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

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

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swbarnes2 ★ 1.4k
@swbarnes2-14086
Last seen 10 hours 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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