Greetings,

Until November 2025, Guitar was working OK for me. Now (December 2025) when I try to execute GuitarPlot(), providing a GTF file in the txGTF argument, like this:

GuitarPlot(txGTF = "REFERENCE.gtf", 
                 stBedFiles = "REGIONS.bed",
                 pltTxType = "mrna", 
                 enableCI = FALSE, 
                headOrtail = FALSE, 
                stGroupName = c("ALL"))

The following error is raised:

Error in call_fun_in_txdbmaker("makeTxDbFromGFF", ...) : 
  makeTxDbFromGFF() has moved from GenomicFeatures to the txdbmaker package, and is formally defunct in GenomicFeatures >= 1.61.1. Please call
  txdbmaker::makeTxDbFromGFF() to get rid of this error.

According to this error the function makeTxDbFromGFF() (which is used internally by GuitarPlot()) now belongs to the package txdbmaker. This causes a conflict because the version of GenomicFeatures provided by Bioconductor 3.22 is 1.62.0. Probably downgrading to an earlier version of Bioconductor (where GenomicFeatures::makeTxDbFromGFF() existed will work as a temporary fix for this issue.

In my case, I fixed the issue by:

1- Cloning the source repository from Bioconductor

git clone https://git.bioconductor.org/packages/Guitar

2- Changing the following line of code

• Line 79 of Guitar/R/GuiatrPlot.R

txdb <- txdbmaker::makeTxDbFromGFF(file=txGTF, format="gtf")

Due to this manual edits in the source files, some dependencies were not properly loaded, personally I had issues with the reduce function from GenomicRanges, having to specify the source package when called (in any of the scripts located in Guitar/R): GenomicRanges:reduce().

3- Load all the R scripts belonging to Guitar, located in Guitar/R:

# Unmodified scripts
 source("/home/fllobet/m6A-Seq/LIVER/Guitar/R/siteNormalize.R")
source("/home/fllobet/m6A-Seq/LIVER/Guitar/R/sitesProcess.R")
# Modified scripts
source("/home/fllobet/m6A-Seq/LIVER/Guitar/R/GuitarPlot.R")
source("/home/fllobet/m6A-Seq/LIVER/Guitar/R/GenomicRangesOps.R")
source("/home/fllobet/m6A-Seq/LIVER/Guitar/R/makeGuitarTxdb.R")

After this changes, execution of GuitarPlot using the arguments above produces the expected plot:

GuitarPlot(txGTF = "REFERENCE.gtf", 
+            stBedFiles = "DMP.bed",
+            pltTxType = "mrna", 
+            enableCI = FALSE, 
+            headOrtail = FALSE, 
+            stGroupName = c("DMP"))
[1] "20251229132619"
[1] "get transcriptome annotation from GTF file REFERENCE.gtf"
Import genomic features from the file as a GRanges object ... OK
Prepare the 'metadata' data frame ... OK
Make the TxDb object ... 
Warning 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.
Warning in .makeTxDb_normarg_chrominfo(chrominfo) :
  genome version information is not available for this TxDb object
OK
[1] "There are 60440 transcripts of 35682 genes in the genome."
[1] "total 60440 transcripts extracted ..."
[1] "total 54319 transcripts left after ambiguity filter ..."
[1] "total 54319 transcripts left after check chromosome validity ..."
[1] "total 18708 mRNAs left after component length filter ..."
[1] "total 13635 ncRNAs left after ncRNA length filter ..."
[1] "generate components for mRNA"
[1] "generate chiped transcriptome"
[1] "generate coverage checking ranges for mrna"
[1] "20251229132947"
[1] "import BED file ~/m6A-Seq/LIVER/DMP/3v3/peak_cali_DMP.bed"
[1] "sample 10 points for DMP"
[1] "start figure plotting for mrna ..."
Warning: Using `size` aesthetic for lines was deprecated in ggplot2 3.4.0.
**i** Please use `linewidth` instead.
This warning is displayed once every 8 hours.
Call `lifecycle::last_lifecycle_warnings()` to see where this warning was generated.

I leave this message as quick fix for if anyone else experiments this issue. Hopefully the developers of Guitar can see it and fix the package for everyone. Probably other use-cases of Guitar might raise similar errors.

Thank you,

Ferran.

> sessionInfo()
R version 4.5.2 (2025-10-31)
Platform: x86_64-pc-linux-gnu
Running under: Ubuntu 24.04.3 LTS

Matrix products: default
BLAS:   /opt/R/4.5.2_cairo/lib/R/lib/libRblas.so 
LAPACK: /opt/R/4.5.2_cairo/lib/R/lib/libRlapack.so;  LAPACK version 3.12.1

locale:
 [1] LC_CTYPE=en_US.UTF-8         
 [2] LC_NUMERIC=C                 
 [3] LC_TIME=en_US.UTF-8          
 [4] LC_COLLATE=en_US.UTF-8       
 [5] LC_MONETARY=en_US.UTF-8      
 [6] LC_MESSAGES=en_US.UTF-8      
 [7] LC_PAPER=en_US.UTF-8         
 [8] LC_NAME=en_US.UTF-8          
 [9] LC_ADDRESS=en_US.UTF-8       
[10] LC_TELEPHONE=en_US.UTF-8     
[11] LC_MEASUREMENT=en_US.UTF-8   
[12] LC_IDENTIFICATION=en_US.UTF-8

time zone: Europe/Madrid
tzcode source: system (glibc)

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

other attached packages:
 [1] txdbmaker_1.6.2             magrittr_2.0.4             
 [3] GenomicFeatures_1.62.0      AnnotationDbi_1.72.0       
 [5] GenomeInfoDb_1.46.2         plyranges_1.30.1           
 [7] edgeR_4.8.1                 limma_3.66.0               
 [9] lubridate_1.9.4             forcats_1.0.1              
[11] stringr_1.6.0               dplyr_1.1.4                
[13] purrr_1.2.0                 readr_2.1.6                
[15] tidyr_1.3.2                 tibble_3.3.0               
[17] tidyverse_2.0.0             vsn_3.78.0                 
[19] DESeq2_1.50.2               SummarizedExperiment_1.40.0
[21] Biobase_2.70.0              MatrixGenerics_1.22.0      
[23] matrixStats_1.5.0           circlize_0.4.17            
[25] svglite_2.2.2               rtracklayer_1.70.1         
[27] GenomicRanges_1.62.1        Seqinfo_1.0.0              
[29] IRanges_2.44.0              S4Vectors_0.48.0           
[31] ggbio_1.58.0                BiocGenerics_0.56.0        
[33] generics_0.1.4              ggplot2_4.0.1              

loaded via a namespace (and not attached):
  [1] BiocIO_1.20.0            filelock_1.0.3          
  [3] bitops_1.0-9             cellranger_1.1.0        
  [5] preprocessCore_1.72.0    graph_1.88.1            
  [7] XML_3.99-0.20            rpart_4.1.24            
  [9] httr2_1.2.2              lifecycle_1.0.4         
 [11] lattice_0.22-7           vroom_1.6.7             
 [13] ensembldb_2.34.0         OrganismDbi_1.52.0      
 [15] backports_1.5.0          Hmisc_5.2-4             
 [17] rmarkdown_2.30           yaml_2.3.12             
 [19] otel_0.2.0               DBI_1.2.3               
 [21] RColorBrewer_1.1-3       Guitar_2.26.0           
 [23] abind_1.4-8              AnnotationFilter_1.34.0 
 [25] biovizBase_1.58.0        RCurl_1.98-1.17         
 [27] nnet_7.3-20              rappdirs_0.3.3          
 [29] VariantAnnotation_1.56.0 xlsxjars_0.9.0          
 [31] ggrepel_0.9.6            codetools_0.2-20        
 [33] DelayedArray_0.36.0      tidyselect_1.2.1        
 [35] shape_1.4.6.1            UCSC.utils_1.6.1        
 [37] farver_2.1.2             BiocFileCache_3.0.0     
 [39] base64enc_0.1-3          GenomicAlignments_1.46.0
 [41] jsonlite_2.0.0           Formula_1.2-5           
 [43] systemfonts_1.3.1        progress_1.2.3          
 [45] tools_4.5.2              ragg_1.5.0              
 [47] Rcpp_1.1.0               glue_1.8.0              
 [49] gridExtra_2.3            SparseArray_1.10.8      
 [51] xfun_0.55                withr_3.0.2             
 [53] BiocManager_1.30.27      fastmap_1.2.0           
 [55] digest_0.6.39            timechange_0.3.0        
 [57] R6_2.6.1                 textshaping_1.0.4       
 [59] colorspace_2.1-2         rsvg_2.7.0              
 [61] dichromat_2.0-0.1        biomaRt_2.66.0          
 [63] RSQLite_2.4.5            cigarillo_1.0.0         
 [65] hexbin_1.28.5            data.table_1.18.0       
 [67] prettyunits_1.2.0        httr_1.4.7              
 [69] htmlwidgets_1.6.4        S4Arrays_1.10.1         
 [71] pkgconfig_2.0.3          rJava_1.0-11            
 [73] gtable_0.3.6             blob_1.2.4              
 [75] S7_0.2.1                 XVector_0.50.0          
 [77] htmltools_0.5.9          RBGL_1.86.0             
 [79] ProtGenerics_1.42.0      scales_1.4.0            
 [81] png_0.1-8                knitr_1.51              
 [83] rstudioapi_0.17.1        tzdb_0.5.0              
 [85] reshape2_1.4.5           rjson_0.2.23            
 [87] checkmate_2.3.3          curl_7.0.0              
 [89] cachem_1.1.0             GlobalOptions_0.1.3     
 [91] parallel_4.5.2           foreign_0.8-90          
 [93] restfulr_0.0.16          pillar_1.11.1           
 [95] grid_4.5.2               vctrs_0.6.5             
 [97] dbplyr_2.5.1             cluster_2.1.8.1         
 [99] htmlTable_2.4.3          evaluate_1.0.5          
[101] cli_3.6.5                locfit_1.5-9.12         
[103] compiler_4.5.2           Rsamtools_2.26.0        
[105] rlang_1.1.6              crayon_1.5.3            
[107] labeling_0.4.3           affy_1.88.0             
[109] plyr_1.8.9               stringi_1.8.7           
[111] BiocParallel_1.44.0      Biostrings_2.78.0       
[113] lazyeval_0.2.2           Matrix_1.7-4            
[115] BSgenome_1.78.0          hms_1.1.4               
[117] bit64_4.6.0-1            KEGGREST_1.50.0         
[119] statmod_1.5.1            memoise_2.0.1           
[121] affyio_1.80.0            bit_4.6.0               
[123] readxl_1.4.5