All samples have 0 counts for all genes. check the counting script

@2f3f6904

Last seen 1 hour 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.

@swbarnes2-14086

Last seen 15 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.