IsoformSwitchAnalyzeR Error in reading GTF file

Question

[VenGeno]

Hi,

I am using IsoformSwitchAnalyzeR v1.21.0 in R 4.3.0 environment using RStudio (2023.06.0+421 "Mountain Hydrangea" Release). When I try to import GTF file (obtained from Ensembl Plants) I get the following error ;

Step 1 of 7: Checking data... Step 2 of 7: Obtaining annotation... importing GTF (this may take a while)... Error in dplyr::full_join(): ! ... must be empty.

I was using the following code;

### Create switchAnalyzeRlist
aSwitchList <- importRdata(
  isoformCountMatrix   = KallistoQuant$counts,
  isoformRepExpression = KallistoQuant$abundance,
  designMatrix         = myDesign,
  isoformExonAnnoation = "Arabidopsis_thaliana.TAIR10.53.gtf.gz",
  isoformNtFasta       = "Arabidopsis_thaliana.TAIR10.cdna.all.fa",
  comparisonsToMake = cond,
  showProgress = FALSE
)

Can anyone help me with this error? I have run this annotation file and fasta earlier without any issues (a year back).

Thank you in advance.

Venura

sessionInfo( )

R version 4.3.0 (2023-04-21 ucrt) Platform: x86_64-w64-mingw32/x64 (64-bit) Running under: Windows 10 x64 (build 19045)

Matrix products: default

locale: [1] LC_COLLATE=English_United States.utf8 LC_CTYPE=English_United States.utf8 LC_MONETARY=English_United States.utf8 LC_NUMERIC=C
[5] LC_TIME=English_United States.utf8

time zone: Asia/Colombo tzcode source: internal

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

other attached packages: [1] IsoformSwitchAnalyzeR_1.21.0 ggplot2_3.4.2 DEXSeq_1.46.0 RColorBrewer_1.1-3 AnnotationDbi_1.63.1
[6] DESeq2_1.41.2 SummarizedExperiment_1.31.1 GenomicRanges_1.53.1 GenomeInfoDb_1.37.2 IRanges_2.35.2
[11] S4Vectors_0.39.1 MatrixGenerics_1.13.0 matrixStats_1.0.0 Biobase_2.61.0 BiocGenerics_0.47.0
[16] BiocParallel_1.35.2 limma_3.57.6 BiocManager_1.30.21

loaded via a namespace (and not attached): [1] rstudioapi_0.14 jsonlite_1.8.5 tximport_1.29.0 magrittr_2.0.3 GenomicFeatures_1.53.1
[6] BiocIO_1.11.0 zlibbioc_1.47.0 vctrs_0.6.3 memoise_2.0.1 Rsamtools_2.17.0
[11] RCurl_1.98-1.12 htmltools_0.5.5 S4Arrays_1.1.4 progress_1.2.2 AnnotationHub_3.9.1
[16] lambda.r_1.2.4 curl_5.0.1 Rhdf5lib_1.23.0 rhdf5_2.45.0 SparseArray_1.1.10
[21] plyr_1.8.8 futile.options_1.0.1 cachem_1.0.8 GenomicAlignments_1.37.0 mime_0.12
[26] lifecycle_1.0.3 pkgconfig_2.0.3 Matrix_1.5-4.1 R6_2.5.1 fastmap_1.1.1
[31] GenomeInfoDbData_1.2.10 shiny_1.7.4 digest_0.6.31 colorspace_2.1-0 tximeta_1.19.0
[36] geneplotter_1.79.1 RSQLite_2.3.1 hwriter_1.3.2.1 filelock_1.0.2 fansi_1.0.4
[41] httr_1.4.6 compiler_4.3.0 bit64_4.0.5 withr_2.5.0 DBI_1.1.3
[46] biomaRt_2.57.1 rappdirs_0.3.3 DelayedArray_0.27.5 rjson_0.2.21 tools_4.3.0
[51] interactiveDisplayBase_1.39.0 DRIMSeq_1.29.0 httpuv_1.6.11 glue_1.6.2 VennDiagram_1.7.3
[56] restfulr_0.0.15 rhdf5filters_1.13.3 promises_1.2.0.1 grid_4.3.0 reshape2_1.4.4
[61] generics_0.1.3 gtable_0.3.3 BSgenome_1.69.0 tzdb_0.4.0 ensembldb_2.25.0
[66] hms_1.1.3 xml2_1.3.4 utf8_1.2.3 XVector_0.41.1 BiocVersion_3.18.0
[71] pillar_1.9.0 stringr_1.5.0 vroom_1.6.3 genefilter_1.83.1 later_1.3.1
[76] splines_4.3.0 dplyr_1.1.2 BiocFileCache_2.9.0 lattice_0.21-8 survival_3.5-5
[81] rtracklayer_1.61.0 bit_4.0.5 annotate_1.79.0 tidyselect_1.2.0 locfit_1.5-9.8
[86] Biostrings_2.69.1 gridExtra_2.3 ProtGenerics_1.33.1 edgeR_3.43.7 futile.logger_1.4.3
[91] statmod_1.5.0 stringi_1.7.12 lazyeval_0.2.2 yaml_2.3.7 codetools_0.2-19
[96] tibble_3.2.1 cli_3.6.1 xtable_1.8-4 munsell_0.5.0 Rcpp_1.0.10
[101] dbplyr_2.3.2 png_0.1-8 XML_3.99-0.14 parallel_4.3.0 ellipsis_0.3.2
[106] readr_2.1.4 blob_1.2.4 prettyunits_1.1.1 AnnotationFilter_1.25.0 bitops_1.0-7
[111] scales_1.2.1 crayon_1.5.2 rlang_1.1.1 KEGGREST_1.41.0 formatR_1.14

[Trivas]

Does this error persist when you gunzip your GTF file?

[VenGeno]

yes the same error appears.

[Trivas]

My guess is that you are using cDNA sequence and not the unprocessed mRNA transcript.

Answer 1

[VenGeno]

Hi All,

The error was version related. It was resolved once the developer version (instead of Bioconductor version) was installed using the following script.

if (!requireNamespace("devtools", quietly = TRUE)){
    install.packages("devtools")
}
devtools::install_github("kvittingseerup/IsoformSwitchAnalyzeR", build_vignettes = TRUE)

Thank you for all the assistance.