Last updated: 2020-04-28

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Knit directory: BgeeCall_practical/

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Introduction

This page describe how to generate present/absent calls for one RNA-Seq library.

BgeeMetadata and KallistoMetadata objects

These 2 classes are useful to tune how BgeeCall works. No need to create an object of the class BgeeMetada as only default values will be used during this practical (last official Bgee intergenic release).
An object of the KallistoMetadata class has to be created to specify to download kallisto (as it is not installed on RStudio cloud).

kallisto <- new("KallistoMetadata", download_kallisto = TRUE)

UserMetadata object

An object of this class has to be created and value of some slots have to be modified to run BgeeCall

user <- new("UserMetadata", species_id = "NCBI_TAXON_ID", reads_size=READS_SIZE)
user <- setRNASeqLibPath(user, "PATH_TO_RNASEQ_LIBRARY_DIR")
user <- setTranscriptomeFromFile(user, "PATH_TO_TRANSCRIPTOME")
user <- setAnnotationFromFile(user, "PATH_TO_GTF_FILE")
user <- setOutputDir(user, "PATH_TO_OUTPUT_DIR")
user <- setWorkingPath(user, "PATH_TO_WORKING_DIR")

Generate present/absent calls

Now that all objects have been created it is possible to run the generation of present/absent gene expression calls with one unique line of code

output_files_info <- generate_calls_workflow(abundanceMetadata = kallisto, userMetadata = user)
output_files_info
$calls_tsv_path
[1] "PATH_TO_OUTPUT_DIR/gene_level_abundance+calls.tsv"

$cutoff_info_file_path
[1] "PATH_TO_OUTPUT_DIR/gene_cutoff_info_file.tsv"

$abundance_tsv
[1] "PATH_TO_OUTPUT_DIR/abundance.tsv"

$TPM_distribution_path
[1] "PATH_TO_OUTPUT_DIR/gene_TPM_genic_intergenic+cutoff.pdf"

$S4_slots_summary
[1] "PATH_TO_OUTPUT_DIR/S4_slots_summary.tsv"

This command generates a list with a link to 5 generated files.

  • calls_tsv_path : path to the file with abundance at gene level and present/absent expression calls
  • cutoff_info_file_path : path to the file with metadata about TPM cutoff (TPM cutoff, protein coding genes present, etc.)
  • abundance_tsv : path to the kallisto output file with abundance at transcript level
  • TPM_distribution_path : path to a PDF file containing a plot of abundance distribution with a line representing the TPM cutoff.
  • S4_slots_sumary : path to the file with value of most important slots of the 3 BgeeCall classes

sessionInfo()
R version 3.6.3 (2020-02-29)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Ubuntu 18.04.4 LTS

Matrix products: default
BLAS:   /usr/lib/x86_64-linux-gnu/openblas/libblas.so.3
LAPACK: /usr/lib/x86_64-linux-gnu/libopenblasp-r0.2.20.so

locale:
 [1] LC_CTYPE=en_GB.UTF-8       LC_NUMERIC=C              
 [3] LC_TIME=fr_FR.UTF-8        LC_COLLATE=en_GB.UTF-8    
 [5] LC_MONETARY=fr_FR.UTF-8    LC_MESSAGES=en_GB.UTF-8   
 [7] LC_PAPER=fr_FR.UTF-8       LC_NAME=C                 
 [9] LC_ADDRESS=C               LC_TELEPHONE=C            
[11] LC_MEASUREMENT=fr_FR.UTF-8 LC_IDENTIFICATION=C       

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

other attached packages:
[1] BgeeCall_1.2.1  workflowr_1.6.1

loaded via a namespace (and not attached):
 [1] Rcpp_1.0.3                  lattice_0.20-41            
 [3] prettyunits_1.1.1           Rsamtools_2.2.3            
 [5] Biostrings_2.54.0           assertthat_0.2.1           
 [7] rprojroot_1.3-2             digest_0.6.25              
 [9] BiocFileCache_1.10.2        R6_2.4.1                   
[11] GenomeInfoDb_1.22.0         backports_1.1.5            
[13] stats4_3.6.3                RSQLite_2.2.0              
[15] evaluate_0.14               httr_1.4.1                 
[17] pillar_1.4.3                zlibbioc_1.32.0            
[19] rlang_0.4.5                 GenomicFeatures_1.38.2     
[21] progress_1.2.2              curl_4.3                   
[23] whisker_0.4                 blob_1.2.1                 
[25] S4Vectors_0.24.3            Matrix_1.2-18              
[27] rmarkdown_2.1               BiocParallel_1.20.1        
[29] stringr_1.4.0               RCurl_1.98-1.1             
[31] bit_1.1-15.2                biomaRt_2.42.0             
[33] DelayedArray_0.12.2         compiler_3.6.3             
[35] httpuv_1.5.2                rtracklayer_1.46.0         
[37] xfun_0.12                   pkgconfig_2.0.3            
[39] askpass_1.1                 BiocGenerics_0.32.0        
[41] htmltools_0.4.0             tximport_1.14.0            
[43] openssl_1.4.1               tidyselect_1.0.0           
[45] SummarizedExperiment_1.16.1 tibble_2.1.3               
[47] GenomeInfoDbData_1.2.2      matrixStats_0.55.0         
[49] IRanges_2.20.2              XML_3.99-0.3               
[51] crayon_1.3.4                dplyr_0.8.4                
[53] dbplyr_1.4.2                later_1.0.0                
[55] GenomicAlignments_1.22.1    bitops_1.0-6               
[57] rappdirs_0.3.1              grid_3.6.3                 
[59] jsonlite_1.6.1              DBI_1.1.0                  
[61] git2r_0.26.1                magrittr_1.5               
[63] stringi_1.4.6               XVector_0.26.0             
[65] fs_1.3.2                    promises_1.1.0             
[67] vctrs_0.2.3                 Rhdf5lib_1.8.0             
[69] tools_3.6.3                 bit64_0.9-7                
[71] Biobase_2.46.0              glue_1.3.1                 
[73] purrr_0.3.3                 hms_0.5.3                  
[75] parallel_3.6.3              yaml_2.2.1                 
[77] rhdf5_2.30.1                AnnotationDbi_1.48.0       
[79] GenomicRanges_1.38.0        memoise_1.1.0              
[81] knitr_1.28