Error generating counts df for use with DRIMSeq/DEXseq
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jbono • 0
@jbono-7682
Last seen 17 hours ago
United States

Hi,

I am attempting to work through the workflow described in "Swimming downstream: statistical analysis of differential transcript usage following Salmon quantification." I am running into an error message when I try to make the counts dataframe for DRIMseq: Error in data.frame(gene_id = txdf$GENEID, feature_id = txdf$TXNAME, cts) : arguments imply differing number of rows: 35367, 28234

I assume this means there are transcripts in the counts object (cts) I generated from salmon that are not present in the txdb. I'm not sure if this is related to some warning messages I got during the construction of the txdb (see below) or something else, but I'm not sure what I would need to do to fix the problem. Any help would be much appreciated! Thanks.

> sample_information=read.csv("sample_information.csv")
> files <- file.path("salmon/transcripts_quant", sample_information$sample_id, "quant.sf")
> names(files) <- sample_information$sample_id
> txi <- tximport(files, type="salmon", txOut=TRUE,
+                 countsFromAbundance="scaledTPM")
reading in files with read_tsv
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 
> cts <- txi$counts
> cts <- cts[rowSums(cts) > 0,]
> library(GenomicFeatures)
> Dmel_r6.38_gtf <- "dmel-all-r6.38.gtf.gz"
> txdb.filename <- "Dmel_r6.38_gtf.sqlite"
> txdb <- makeTxDbFromGFF(Dmel_r6.38_gtf)
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... OK
Warning messages:
1: In .get_cds_IDX(mcols0$type, mcols0$phase) :
  The "phase" metadata column contains non-NA values for features of type stop_codon. This information was
  ignored.
2: In makeTxDbFromGRanges(gr, metadata = metadata) :
  The following transcripts were dropped because their exon ranks could not be inferred (either because the
  exons are not on the same chromosome/strand or because they are not separated by introns): FBtr0084079,
  FBtr0084080, FBtr0084081, FBtr0084082, FBtr0084083, FBtr0084084, FBtr0084085, FBtr0307759, FBtr0307760
3: In .reject_transcripts(bad_tx, because) :
  The following transcripts were rejected because they have stop codons that cannot be mapped to an exon:
  FBtr0100857, FBtr0100863, FBtr0433500, FBtr0433501
> saveDb(txdb, txdb.filename)
TxDb object:
# Db type: TxDb
# Supporting package: GenomicFeatures
# Data source: dmel-all-r6.38.gtf.gz
# Organism: NA
# Taxonomy ID: NA
# miRBase build ID: NA
# Genome: NA
# Nb of transcripts: 35367
# Db created by: GenomicFeatures package from Bioconductor
# Creation time: 2021-04-05 12:20:51 -0600 (Mon, 05 Apr 2021)
# GenomicFeatures version at creation time: 1.42.3
# RSQLite version at creation time: 2.2.5
# DBSCHEMAVERSION: 1.2
> txdf <- select(txdb, keys(txdb, "GENEID"), "TXNAME", "GENEID")
'select()' returned 1:many mapping between keys and columns
> tab <- table(txdf$GENEID)
> txdf$ntx <- tab[match(txdf$GENEID, names(tab))]
> dtu_counts <- data.frame(gene_id=txdf$GENEID,
+                          feature_id=txdf$TXNAME,
+                          cts)
Error in data.frame(gene_id = txdf$GENEID, feature_id = txdf$TXNAME, cts) : 
  arguments imply differing number of rows: 35367, 28234

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

> sessionInfo()
R version 4.0.4 (2021-02-15)
Platform: x86_64-apple-darwin17.0 (64-bit)
Running under: macOS Catalina 10.15.7

Matrix products: default
BLAS:   /System/Library/Frameworks/Accelerate.framework/Versions/A/Frameworks/vecLib.framework/Versions/A/libBLAS.dylib
LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib

locale:
[1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8

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

other attached packages:
 [1] tximport_1.18.0              IsoformSwitchAnalyzeR_1.12.0 ggplot2_3.3.3                DEXSeq_1.36.0               
 [5] RColorBrewer_1.1-2           DESeq2_1.30.1                BiocParallel_1.24.1          GenomicFeatures_1.42.3      
 [9] GenomicAlignments_1.26.0     Rsamtools_2.6.0              Biostrings_2.58.0            XVector_0.30.0              
[13] SummarizedExperiment_1.20.0  MatrixGenerics_1.2.1         matrixStats_0.58.0           GenomicRanges_1.42.0        
[17] GenomeInfoDb_1.26.4          GenomeInfoDbData_1.2.4       ASpli_2.0.0                  AnnotationDbi_1.52.0        
[21] IRanges_2.24.1               S4Vectors_0.28.1             Biobase_2.50.0               BiocGenerics_0.36.0         
[25] Rsubread_2.4.3               edgeR_3.32.1                 limma_3.46.0                

loaded via a namespace (and not attached):
  [1] backports_1.2.1               AnnotationHub_2.22.0          Hmisc_4.5-0                  
  [4] DRIMSeq_1.18.0                BiocFileCache_1.14.0          plyr_1.8.6                   
  [7] igraph_1.2.6                  lazyeval_0.2.2                tximeta_1.8.4                
 [10] splines_4.0.4                 digest_0.6.27                 ensembldb_2.14.0             
 [13] htmltools_0.5.1.1             fansi_0.4.2                   magrittr_2.0.1               
 [16] checkmate_2.0.0               memoise_2.0.0                 BSgenome_1.58.0              
 [19] cluster_2.1.1                 readr_1.4.0                   annotate_1.68.0              
 [22] askpass_1.1                   prettyunits_1.1.1             jpeg_0.1-8.1                 
 [25] colorspace_2.0-0              blob_1.2.1                    rappdirs_0.3.3               
 [28] xfun_0.22                     dplyr_1.0.5                   jsonlite_1.7.2               
 [31] crayon_1.4.1                  RCurl_1.98-1.3                genefilter_1.72.1            
 [34] survival_3.2-10               VariantAnnotation_1.36.0      glue_1.4.2                   
 [37] gtable_0.3.0                  zlibbioc_1.36.0               UpSetR_1.4.0                 
 [40] DelayedArray_0.16.3           scales_1.1.1                  futile.options_1.0.1         
 [43] DBI_1.1.1                     Rcpp_1.0.6                    xtable_1.8-4                 
 [46] progress_1.2.2                htmlTable_2.1.0               foreign_0.8-81               
 [49] bit_4.0.4                     Formula_1.2-4                 DT_0.17                      
 [52] htmlwidgets_1.5.3             httr_1.4.2                    ellipsis_0.3.1               
 [55] pkgconfig_2.0.3               XML_3.99-0.6                  Gviz_1.34.1                  
 [58] nnet_7.3-15                   dbplyr_2.1.0                  locfit_1.5-9.4               
 [61] utf8_1.2.1                    later_1.1.0.1                 tidyselect_1.1.0             
 [64] rlang_0.4.10                  reshape2_1.4.4                BiocVersion_3.12.0           
 [67] munsell_0.5.0                 tools_4.0.4                   cachem_1.0.4                 
 [70] generics_0.1.0                RSQLite_2.2.5                 evaluate_0.14                
 [73] stringr_1.4.0                 fastmap_1.1.0                 yaml_2.2.1                   
 [76] knitr_1.31                    bit64_4.0.5                   purrr_0.3.4                  
 [79] AnnotationFilter_1.14.0       mime_0.10                     formatR_1.8                  
 [82] xml2_1.3.2                    biomaRt_2.46.3                BiocStyle_2.18.1             
 [85] compiler_4.0.4                rstudioapi_0.13               interactiveDisplayBase_1.28.0
 [88] curl_4.3                      png_0.1-7                     tibble_3.1.0                 
 [91] statmod_1.4.35                geneplotter_1.68.0            stringi_1.5.3                
 [94] futile.logger_1.4.3           lattice_0.20-41               ProtGenerics_1.22.0          
 [97] Matrix_1.3-2                  vctrs_0.3.7                   pillar_1.5.1                 
[100] lifecycle_1.0.0               BiocManager_1.30.12           data.table_1.14.0            
[103] bitops_1.0-6                  httpuv_1.5.5                  rtracklayer_1.50.0           
[106] R6_2.5.0                      latticeExtra_0.6-29           hwriter_1.3.2                
[109] promises_1.2.0.1              gridExtra_2.3                 lambda.r_1.2.4               
[112] dichromat_2.0-0               MASS_7.3-53.1                 assertthat_0.2.1             
[115] openssl_1.4.3                 withr_2.4.1                   hms_1.0.0                    
[118] VennDiagram_1.6.20            grid_4.0.4                    rpart_4.1-15                 
[121] tidyr_1.1.3                   rmarkdown_2.7                 biovizBase_1.38.0            
[124] shiny_1.6.0                   base64enc_0.1-3               tinytex_0.31
DRIMSeq DEXSeq • 42 views
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