Last updated: 2024-10-09

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Knit directory: GibbsDTE-code/analysis/

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Note that any generated files, e.g. HTML, png, CSS, etc., are not included in this status report because it is ok for generated content to have uncommitted changes.

These are the previous versions of the repository in which changes were made to the R Markdown (analysis/lung-howto.Rmd) and HTML (docs/lung-howto.html) files. If you’ve configured a remote Git repository (see ?wflow_git_remote), click on the hyperlinks in the table below to view the files as they were in that past version.

File	Version	Author	Date	Message
html	70ab63f	Pedro Baldoni	2024-04-05	Publishing workflowr
Rmd	afc521e	Pedro Baldoni	2024-04-05	Adding case study dataset Rmd

Our case study is based on the paired-end Illumina short read RNA-seq experiment from the human lung cancer cell lines H1975 and HCC827 (GSE172421).

All the RNA-seq experiments were quantified with Salmon version v1.10.0 using an decoy-aware indexed transcriptome based on the Ensembl-oriented Gencode annotation version 33 (Ensembl annotation version 99) with added transcripts from spiked-in sequins. We also quantified the RNA-seq experiments with kallisto version v0.46.1 using an indexed transcriptome based on the Gencode annotation version 33 to assess the consistency of RTA overdispersion estimates in edgeR. To generate coverage plots, we have aligned RNA-seq experiments to the hg38 reference genome with added spiked-in sequins genome with Subread.

Acessing the necessary annotation

Download the Gencode annotation version 33 (Ensembl annotation version 99) from this link and place files in the directory ./data/annotation/hg38. Files can be downloaded with the following command line: wget -r -nd ftp://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_human/release_33/*

The sequins annotation is located in the directory ./data/annotation/sequins of the GitHub repository of this page. They were originally obtained from this repo.

The RefSeq gene annotation used by Subread during alignment is located in the directory ./data/annotation/hg38 of the GitHub repository of this page. This annotation was originally obtained from the curated annotation database for Rsubread that is actively maintained by WEHI Bioinformatics.

Creating the reference genome and transcriptome

Run the script code/lung/index/combineAnnotation.sh to combine the Gencode annotation with the sequins annotation. This script uses the picard-tools module.
Run the script code/lung/index/buildIndex.sh to build Salmon’s transcriptome index. This script uses the following modules: Salmon, MashMap, bedtools and the generateDecoyTranscriptome.sh script.
Run the script code/lung/index/index-kallisto/slurm.sh to build kallisto’s transcriptome index.
Run the script code/lung/index-subread to build the Subread index. This script uses the combined reference genome created in step 1.

Downloading, quantifying, and aligning the Illumina paired-end short read experiments

Run ./code/lung/data/runAspera.sh to download all necessary FASTQ files. This script uses the module aspera.
Run ./code/lung/salmon/runSalmon.sh to quantify the RNA-seq reads with Salmon.
Run ./code/lung/salmon/runWasabi.R to generate .h5 files from Salmon’s output that can be read by the DTE method sleuth.
Run the script ./code/lung/kallisto/runKallisto.sh to quantify the RNA-seq reads with kallisto.
Run the script ./code/lung/subread/slurm.sh to align the RNA-seq samples with Subread.

The targets file for this experiment is provided in our GitHub repository for convenience. See file ./data/lung/misc/targets.txt.

sessionInfo()

R version 4.3.3 (2024-02-29)
Platform: x86_64-pc-linux-gnu (64-bit)
Running under: Red Hat Enterprise Linux 9.4 (Plow)

Matrix products: default
BLAS:   /stornext/System/data/software/rhel/9/base/tools/R/4.3.3/lib64/R/lib/libRblas.so 
LAPACK: /stornext/System/data/software/rhel/9/base/tools/R/4.3.3/lib64/R/lib/libRlapack.so;  LAPACK version 3.11.0

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

time zone: Australia/Melbourne
tzcode source: system (glibc)

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

other attached packages:
[1] workflowr_1.7.1

loaded via a namespace (and not attached):
 [1] jsonlite_1.8.9      compiler_4.3.3      BiocManager_1.30.25
 [4] renv_1.0.9          promises_1.3.0      Rcpp_1.0.13        
 [7] stringr_1.5.1       git2r_0.33.0        callr_3.7.6        
[10] later_1.3.2         jquerylib_0.1.4     yaml_2.3.10        
[13] fastmap_1.2.0       R6_2.5.1            knitr_1.48         
[16] tibble_3.2.1        rprojroot_2.0.4     bslib_0.8.0        
[19] pillar_1.9.0        rlang_1.1.4         utf8_1.2.4         
[22] cachem_1.1.0        stringi_1.8.4       httpuv_1.6.15      
[25] xfun_0.47           getPass_0.2-4       fs_1.6.4           
[28] sass_0.4.9          cli_3.6.3           magrittr_2.0.3     
[31] ps_1.8.0            digest_0.6.37       processx_3.8.4     
[34] rstudioapi_0.16.0   lifecycle_1.0.4     vctrs_0.6.5        
[37] evaluate_1.0.0      glue_1.8.0          whisker_0.4.1      
[40] fansi_1.0.6         rmarkdown_2.28      httr_1.4.7         
[43] tools_4.3.3         pkgconfig_2.0.3     htmltools_0.5.8.1

Pre-processing the human lung cancer cell lines dataset

Pedro L. Baldoni

09 October, 2024

Acessing the necessary annotation

Creating the reference genome and transcriptome

Downloading, quantifying, and aligning the Illumina paired-end short read experiments