BgeeDB is a collection of functions to import data from the Bgee database (http://bgee.org/) directly into R, and to facilitate downstream analyses, such as gene set enrichment test based on expression of genes in anatomical structures. Bgee provides annotated and processed expression data and expression calls from curated wild-type healthy samples, from human and many other animal species.
The package retrieves the annotation of RNA-seq or Affymetrix experiments integrated into the Bgee database, and downloads into R the quantitative data and expression calls produced by the Bgee pipeline. The package also allows to run GO-like enrichment analyses based on anatomical terms, where genes are mapped to anatomical terms by expression patterns, based on the topGO package. This is the same as the TopAnat web-service available at (http://bgee.org/?page=top_anat#/), but with more flexibility in the choice of parameters and developmental stages.
In summary, the BgeeDB package allows to: * 1. List annotation of RNA-seq and microarray data available the Bgee database * 2. Download the processed gene expression data available in the Bgee database * 3. Download the gene expression calls and use them to perform TopAnat analyses
In R:
The listBgeeSpecies() function allows to retrieve available species in the Bgee database, and which data types are available for each species.
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## Querying Bgee to get release information...
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## Building URL to query species in Bgee release 14...
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## Submitting URL to Bgee webservice... (http://bgee.org/?page=r_package&action=get_all_species&display_type=tsv&source=BgeeDB_R_package&source_version=2.5.0)
##
## Query to Bgee webservice successful!## ID GENUS SPECIES_NAME COMMON_NAME AFFYMETRIX
## 1 6239 Caenorhabditis elegans nematode TRUE
## 2 7217 Drosophila ananassae FALSE
## 3 7227 Drosophila melanogaster fruit fly TRUE
## 4 7230 Drosophila mojavensis FALSE
## 5 7237 Drosophila pseudoobscura FALSE
## 6 7240 Drosophila simulans FALSE
## 7 7244 Drosophila virilis FALSE
## 8 7245 Drosophila yakuba FALSE
## 9 7955 Danio rerio zebrafish TRUE
## 10 8364 Xenopus tropicalis western clawed frog FALSE
## 11 9031 Gallus gallus chicken FALSE
## 12 9258 Ornithorhynchus anatinus platypus FALSE
## 13 9365 Erinaceus europaeus hedgehog FALSE
## 14 9544 Macaca mulatta macaque TRUE
## 15 9593 Gorilla gorilla gorilla FALSE
## 16 9597 Pan paniscus bonobo FALSE
## 17 9598 Pan troglodytes chimpanzee FALSE
## 18 9606 Homo sapiens human TRUE
## 19 9615 Canis lupus familiaris dog FALSE
## 20 9685 Felis catus cat FALSE
## 21 9796 Equus caballus horse FALSE
## 22 9823 Sus scrofa pig FALSE
## 23 9913 Bos taurus cattle FALSE
## 24 9986 Oryctolagus cuniculus rabbit FALSE
## 25 10090 Mus musculus mouse TRUE
## 26 10116 Rattus norvegicus rat TRUE
## 27 10141 Cavia porcellus guinea pig FALSE
## 28 13616 Monodelphis domestica opossum FALSE
## 29 28377 Anolis carolinensis green anole FALSE
## EST IN_SITU RNA_SEQ
## 1 FALSE TRUE TRUE
## 2 FALSE FALSE TRUE
## 3 TRUE TRUE TRUE
## 4 FALSE FALSE TRUE
## 5 FALSE FALSE TRUE
## 6 FALSE FALSE TRUE
## 7 FALSE FALSE TRUE
## 8 FALSE FALSE TRUE
## 9 TRUE TRUE TRUE
## 10 TRUE TRUE TRUE
## 11 FALSE FALSE TRUE
## 12 FALSE FALSE TRUE
## 13 FALSE FALSE TRUE
## 14 FALSE FALSE TRUE
## 15 FALSE FALSE TRUE
## 16 FALSE FALSE TRUE
## 17 FALSE FALSE TRUE
## 18 TRUE FALSE TRUE
## 19 FALSE FALSE TRUE
## 20 FALSE FALSE TRUE
## 21 FALSE FALSE TRUE
## 22 FALSE FALSE TRUE
## 23 FALSE FALSE TRUE
## 24 FALSE FALSE TRUE
## 25 TRUE TRUE TRUE
## 26 FALSE FALSE TRUE
## 27 FALSE FALSE TRUE
## 28 FALSE FALSE TRUE
## 29 FALSE FALSE TRUEIt is possible to list all species from a specific release of Bgee with the release argument (see listBgeeRelease() function), and order the species according to a specific columns with the ordering argument. For example:
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## Querying Bgee to get release information...
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## Building URL to query species in Bgee release 13_2...
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## Submitting URL to Bgee webservice... (http://bgee.org/bgee13/?page=species&display_type=tsv&source=BgeeDB_R_package&source_version=2.5.0)
##
## Query to Bgee webservice successful!## ID GENUS SPECIES_NAME COMMON_NAME AFFYMETRIX EST IN_SITU
## 17 28377 Anolis carolinensis anolis FALSE FALSE FALSE
## 13 9913 Bos taurus cow FALSE FALSE FALSE
## 1 6239 Caenorhabditis elegans c.elegans TRUE FALSE TRUE
## 3 7955 Danio rerio zebrafish TRUE TRUE TRUE
## 2 7227 Drosophila melanogaster fruitfly TRUE TRUE TRUE
## 5 9031 Gallus gallus chicken FALSE FALSE FALSE
## 8 9593 Gorilla gorilla gorilla FALSE FALSE FALSE
## 11 9606 Homo sapiens human TRUE TRUE FALSE
## 7 9544 Macaca mulatta macaque FALSE FALSE FALSE
## 16 13616 Monodelphis domestica opossum FALSE FALSE FALSE
## 14 10090 Mus musculus mouse TRUE TRUE TRUE
## 6 9258 Ornithorhynchus anatinus platypus FALSE FALSE FALSE
## 9 9597 Pan paniscus bonobo FALSE FALSE FALSE
## 10 9598 Pan troglodytes chimpanzee FALSE FALSE FALSE
## 15 10116 Rattus norvegicus rat FALSE FALSE FALSE
## 12 9823 Sus scrofa pig FALSE FALSE FALSE
## 4 8364 Xenopus tropicalis xenopus FALSE TRUE TRUE
## RNA_SEQ
## 17 TRUE
## 13 TRUE
## 1 TRUE
## 3 FALSE
## 2 FALSE
## 5 TRUE
## 8 TRUE
## 11 TRUE
## 7 TRUE
## 16 TRUE
## 14 TRUE
## 6 TRUE
## 9 TRUE
## 10 TRUE
## 15 TRUE
## 12 TRUE
## 4 TRUEIn the following example we will choose to focus on mouse (“Mus_musculus”) RNA-seq. Species can be specified using their name or their NCBI taxonomic IDs. To specify that RNA-seq data want to be downloaded, the dataType argument is set to “rna_seq”. To download Affymetrix microarray data, set this argument to “affymetrix”.
##
## Querying Bgee to get release information...
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## Building URL to query species in Bgee release 14_0...
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## Submitting URL to Bgee webservice... (http://bgee.org/?page=r_package&action=get_all_species&display_type=tsv&source=BgeeDB_R_package&source_version=2.5.0)
##
## Query to Bgee webservice successful!
##
## API key built: 1a7bb93a14d2d550f3991414b52fe385384cf2e31833ed75cf43b32250dc915a7ba8f5d0fd07b4b79ce2469a5063eeadc17fddff08301180c8df6e131361491eNote 1: It is possible to work with data from a specific release of Bgee by specifying the release argument, see listBgeeRelease() function.
Note 2: The functions of the package will store the downloaded files in a versioned folder created by default in the working directory. These cache files allow faster re-access to the data. The directory where data are stored can be changed with the pathToData argument.
The getAnnotation() function will output the list of RNA-seq experiments and libraries available in Bgee for mouse.
##
## Downloading annotation files...
##
## Saved annotation files in /tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Mus_musculus_Bgee_14_0 folder.## $sample.annotation
## Experiment.ID Library.ID Anatomical.entity.ID Anatomical.entity.name
## 1 GSE30617 ERX012344 UBERON:0000948 heart
## 2 GSE30617 ERX012348 UBERON:0000948 heart
## 3 GSE30617 ERX012362 UBERON:0000948 heart
## 4 GSE30617 ERX012363 UBERON:0000948 heart
## 5 GSE30617 ERX012374 UBERON:0000948 heart
## 6 GSE30617 ERX012378 UBERON:0000948 heart
## Stage.ID Stage.name Sex Strain
## 1 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## 2 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## 3 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## 4 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## 5 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## 6 MmusDv:0000052 8 weeks (mouse) <NA> DBAxC57BL/6J
## Platform.ID Library.type Library.orientation
## 1 Illumina Genome Analyzer IIx paired NA
## 2 Illumina Genome Analyzer IIx paired NA
## 3 Illumina Genome Analyzer IIx paired NA
## 4 Illumina Genome Analyzer IIx paired NA
## 5 Illumina Genome Analyzer IIx paired NA
## 6 Illumina Genome Analyzer IIx paired NA
## TPM.expression.threshold FPKM.expression.threshold Read.count
## 1 0.024357 0.023059 30075234
## 2 0.042566 0.047824 8605668
## 3 0.024598 0.028740 29498377
## 4 0.025378 0.024916 31000737
## 5 0.027712 0.026467 31160789
## 6 0.025381 0.025119 27824366
## Mapped.read.count Min.read.length Max.read.length
## 1 26055677 0 0
## 2 7347266 0 0
## 3 25708709 0 0
## 4 26507831 0 0
## 5 27397595 0 0
## 6 23808859 0 0
## All.genes.percent.present Protein.coding.genes.percent.present
## 1 40.42 69.20
## 2 25.95 51.95
## 3 43.40 69.54
## 4 42.31 70.52
## 5 40.92 68.87
## 6 40.36 68.93
## Intergenic.regions.percent.present Run.IDs Data.source
## 1 14.44 ERR032229 GEO
## 2 6.46 ERR032228 GEO
## 3 17.08 ERR032238 GEO
## 4 15.54 ERR032227 GEO
## 5 14.66 ERR032231 GEO
## 6 14.40 ERR032230 GEO
## Data.source.URL
## 1 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012344
## 2 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012348
## 3 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012362
## 4 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012363
## 5 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012374
## 6 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=ERX012378
## Bgee.normalized.data.URL
## 1 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 2 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 3 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 4 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 5 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 6 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## Raw.file.URL
## 1 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012344
## 2 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012348
## 3 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012362
## 4 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012363
## 5 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012374
## 6 https://trace.ncbi.nlm.nih.gov/Traces/study/?acc=ERX012378
##
## $experiment.annotation
## Experiment.ID
## 1 GSE30617
## 2 GSE41637
## 3 GSE30352
## 4 SRP022612
## 5 GSE36026
## 6 GSE43520
## Experiment.name
## 1 [E-MTAB-599] Mouse Transcriptome
## 2 Evolutionary dynamics of gene and isoform regulation in mammalian tissues
## 3 The evolution of gene expression levels in mammalian organs
## 4 RNA-seq of Danio rerio and Mus musculus skin for three different age groups
## 5 RNA-seq from ENCODE/LICR
## 6 The evolution of lncRNA repertoires and expression patterns in tetrapods
## Library.count Condition.count Organ.stage.count Organ.count Stage.count
## 1 36 6 6 6 1
## 2 26 26 9 9 1
## 3 17 17 6 6 1
## 4 15 2 3 1 3
## 5 12 12 12 12 3
## 6 9 7 5 4 2
## Sex.count Strain.count Data.source
## 1 0 1 GEO
## 2 1 6 GEO
## 3 2 2 GEO
## 4 1 2 GEO
## 5 2 1 GEO
## 6 2 2 GEO
## Data.source.URL
## 1 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30617
## 2 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE41637
## 3 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE30352
## 4 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=SRP022612
## 5 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE36026
## 6 http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE43520
## Bgee.normalized.data.URL
## 1 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip
## 2 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE41637.tsv.zip
## 3 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30352.tsv.zip
## 4 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_SRP022612.tsv.zip
## 5 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE36026.tsv.zip
## 6 ftp://bgee.org/bgee_v14/download/processed_expr_values/rna_seq/Mus_musculus/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE43520.tsv.zip
## Experiment.description
## 1 Sequencing the transcriptome of DBAxC57BL/6J mice. To study the regulation of transcription, splicing and RNA turnover we have sequenced the transcriptomes of tissues collected DBAxC57BL/6J mice.
## 2 Most mammalian genes produce multiple distinct mRNAs through alternative splicing, but the extent of splicing conservation is not clear. To assess tissue-specific transcriptome variation across mammals, we sequenced cDNA from 9 tissues from 4 mammals and one bird in biological triplicate, at unprecedented depth. We find that while tissue-specific gene expression programs are largely conserved, alternative splicing is well conserved in only a subset of tissues and is frequently lineage-specific. Thousands of novel, lineage-specific and conserved alternative exons were identified; widely conserved alternative exons had signatures of binding by MBNL, PTB, RBFOX, STAR and TIA family splicing factors, implicating them as ancestral mammalian splicing regulators. Our data also indicates that alternative splicing is often used to alter protein phosphorylatability, delimiting the scope of kinase signaling.
## 3 Changes in gene expression are thought to underlie many of the phenotypic differences between species. However, large-scale analyses of gene expression evolution were until recently prevented by technological limitations. Here we report the sequencing of polyadenylated RNA from six organs across ten species that represent all major mammalian lineages (placentals, marsupials and monotremes) and birds (the evolutionary outgroup), with the goal of understanding the dynamics of mammalian transcriptome evolution. We show that the rate of gene expression evolution varies among organs, lineages and chromosomes, owing to differences in selective pressures: transcriptome change was slow in nervous tissues and rapid in testes, slower in rodents than in apes and monotremes, and rapid for the X chromosome right after its formation. Although gene expression evolution in mammals was strongly shaped by purifying selection, we identify numerous potentially selectively driven expression switches, which occurred at different rates across lineages and tissues and which probably contributed to the specific organ biology of various mammals. Our transcriptome data provide a valuable resource for functional and evolutionary analyses of mammalian genomes.
## 4 Comparison of temporal gene expression profiles (www.jenage.de) The RNA-seq data comprises 3 age groups: 2, 15 and 30 months for mouse skin; 5, 24 and 42 months for zebrafish skin. Illumina 50bp single-stranded single-read RNA sequencing Overall design: 15 samples for mouse: 5 biological replicates for 2 months, 6 biological replicates for 15 months and 4 biological replicates for 30 months; 20 samples for zebrafish: 9 biological replicates for 5 months, 6 biological replicates for 24 months and 5 biological replicates for 42 months
## 5 Using RNA-Seq (Mortazavi et al., 2008), high-resolution genome-wide maps of the mouse transcriptome across multiple mouse (C57Bl/6) tissues and primary cells were generated.
## 6 To broaden our understanding of lncRNA evolution, we used an extensive RNA-seq dataset to establish lncRNA repertoires and homologous gene families in 11 tetrapod species. We analyzed the poly- adenylated transcriptomes of 8 organs (cortex/whole brain without cerebellum, cerebellum, heart, kidney, liver, placenta, ovary and testis) and 11 species (human, chimpanzee, bonobo, gorilla, orangutan, macaque, mouse, opossum, platypus, chicken and the frog Xenopus tropicalis), which shared a common ancestor ~370 millions of years (MY) ago. Our dataset included 47 strand-specific samples, which allowed us to confirm the orientation of gene predictions and to address the evolution of sense-antisense transcripts. See also GSE43721 (Soumillon et al, Cell Reports, 2013) for three strand-specific samples for mouse brain, liver and testis.The getData() function will download processed RNA-seq data from all mouse experiments in Bgee as a list.
##
## The experiment is not defined. Hence taking all rna_seq experiments available for Mus_musculus.
##
## Downloading expression data...
##
## Saved expression data file in /tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Mus_musculus_Bgee_14_0 folder. Now unzip file...
##
Read 35.5% of 1718244 rows
Read 81.5% of 1718244 rows
Read 1718244 rows and 16 (of 16) columns from 0.284 GB file in 00:00:04
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Read 66.9% of 1240954 rows
Read 1240954 rows and 16 (of 16) columns from 0.213 GB file in 00:00:03
##
## Saving all data in .rds file...## [1] 9## [[1]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE43721 SRX219282 single ENSMUSG00000000001
## 2 GSE43721 SRX219282 single ENSMUSG00000000003
## 3 GSE43721 SRX219282 single ENSMUSG00000000028
## 4 GSE43721 SRX219282 single ENSMUSG00000000031
## 5 GSE43721 SRX219282 single ENSMUSG00000000037
## 6 GSE43721 SRX219282 single ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000473 testis MmusDv:0000055
## 2 UBERON:0000473 testis MmusDv:0000055
## 3 UBERON:0000473 testis MmusDv:0000055
## 4 UBERON:0000473 testis MmusDv:0000055
## 5 UBERON:0000473 testis MmusDv:0000055
## 6 UBERON:0000473 testis MmusDv:0000055
## Stage.name Sex Strain Read.count TPM FPKM
## 1 11 weeks (mouse) male C57BL/6 3077 11.356800 12.591405
## 2 11 weeks (mouse) male C57BL/6 0 0.000000 0.000000
## 3 11 weeks (mouse) male C57BL/6 2491 17.577187 19.488013
## 4 11 weeks (mouse) male C57BL/6 74 0.748961 0.830381
## 5 11 weeks (mouse) male C57BL/6 1659 6.053025 6.711053
## 6 11 weeks (mouse) male C57BL/6 4060 54.832049 60.792873
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[2]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE43013 SRX211656 paired ENSMUSG00000000001
## 2 GSE43013 SRX211656 paired ENSMUSG00000000003
## 3 GSE43013 SRX211656 paired ENSMUSG00000000028
## 4 GSE43013 SRX211656 paired ENSMUSG00000000031
## 5 GSE43013 SRX211656 paired ENSMUSG00000000037
## 6 GSE43013 SRX211656 paired ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 2 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 3 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 4 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 5 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 6 UBERON:0000082 adult mammalian kidney UBERON:0000113
## Stage.name Sex Strain Read.count TPM FPKM
## 1 post-juvenile adult stage male wild-type 1525 46.583577 37.122217
## 2 post-juvenile adult stage male wild-type 0 0.000000 0.000000
## 3 post-juvenile adult stage male wild-type 20 0.889774 0.709056
## 4 post-juvenile adult stage male wild-type 2 0.269064 0.214416
## 5 post-juvenile adult stage male wild-type 5 0.110121 0.087755
## 6 post-juvenile adult stage male wild-type 136 11.615857 9.256618
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[3]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE41338 SRX191152 paired ENSMUSG00000000001
## 2 GSE41338 SRX191152 paired ENSMUSG00000000003
## 3 GSE41338 SRX191152 paired ENSMUSG00000000028
## 4 GSE41338 SRX191152 paired ENSMUSG00000000031
## 5 GSE41338 SRX191152 paired ENSMUSG00000000037
## 6 GSE41338 SRX191152 paired ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 2 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 3 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 4 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 5 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 6 UBERON:0000082 adult mammalian kidney UBERON:0000113
## Stage.name Sex Strain Read.count TPM FPKM
## 1 post-juvenile adult stage mixed C57BL/6 567 14.501564 16.562366
## 2 post-juvenile adult stage mixed C57BL/6 0 0.000000 0.000000
## 3 post-juvenile adult stage mixed C57BL/6 21 0.877042 1.001678
## 4 post-juvenile adult stage mixed C57BL/6 2 0.065568 0.074886
## 5 post-juvenile adult stage mixed C57BL/6 0 0.000000 0.000000
## 6 post-juvenile adult stage mixed C57BL/6 192 12.685089 14.487754
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 absent high quality Part of a call
## 5 absent high quality Part of a call
## 6 present high quality Part of a call
##
## [[4]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE36026 SRX147592 single ENSMUSG00000000001
## 2 GSE36026 SRX147592 single ENSMUSG00000000003
## 3 GSE36026 SRX147592 single ENSMUSG00000000028
## 4 GSE36026 SRX147592 single ENSMUSG00000000031
## 5 GSE36026 SRX147592 single ENSMUSG00000000037
## 6 GSE36026 SRX147592 single ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 CL:0000057 fibroblast MmusDv:0000052
## 2 CL:0000057 fibroblast MmusDv:0000052
## 3 CL:0000057 fibroblast MmusDv:0000052
## 4 CL:0000057 fibroblast MmusDv:0000052
## 5 CL:0000057 fibroblast MmusDv:0000052
## 6 CL:0000057 fibroblast MmusDv:0000052
## Stage.name Sex Strain Read.count TPM FPKM
## 1 8 weeks (mouse) male C57BL/6 5275 26.901318 66.348954
## 2 8 weeks (mouse) male C57BL/6 0 0.000000 0.000000
## 3 8 weeks (mouse) male C57BL/6 380 3.636568 8.969169
## 4 8 weeks (mouse) male C57BL/6 78239 965.590380 2381.515721
## 5 8 weeks (mouse) male C57BL/6 102 0.602173 1.485190
## 6 8 weeks (mouse) male C57BL/6 4 0.109412 0.269851
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[5]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE30617 ERX012344 paired ENSMUSG00000000001
## 2 GSE30617 ERX012344 paired ENSMUSG00000000003
## 3 GSE30617 ERX012344 paired ENSMUSG00000000028
## 4 GSE30617 ERX012344 paired ENSMUSG00000000031
## 5 GSE30617 ERX012344 paired ENSMUSG00000000037
## 6 GSE30617 ERX012344 paired ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000948 heart MmusDv:0000052
## 2 UBERON:0000948 heart MmusDv:0000052
## 3 UBERON:0000948 heart MmusDv:0000052
## 4 UBERON:0000948 heart MmusDv:0000052
## 5 UBERON:0000948 heart MmusDv:0000052
## 6 UBERON:0000948 heart MmusDv:0000052
## Stage.name Sex Strain Read.count TPM FPKM
## 1 8 weeks (mouse) NA DBAxC57BL/6J 1029 14.732067 13.946815
## 2 8 weeks (mouse) NA DBAxC57BL/6J 0 0.000000 0.000000
## 3 8 weeks (mouse) NA DBAxC57BL/6J 61 1.938954 1.835604
## 4 8 weeks (mouse) NA DBAxC57BL/6J 94 3.661912 3.466724
## 5 8 weeks (mouse) NA DBAxC57BL/6J 23 0.212652 0.201317
## 6 8 weeks (mouse) NA DBAxC57BL/6J 8 0.669763 0.634063
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[6]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE43520 SRX217693 single ENSMUSG00000000001
## 2 GSE43520 SRX217693 single ENSMUSG00000000003
## 3 GSE43520 SRX217693 single ENSMUSG00000000028
## 4 GSE43520 SRX217693 single ENSMUSG00000000031
## 5 GSE43520 SRX217693 single ENSMUSG00000000037
## 6 GSE43520 SRX217693 single ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000473 testis UBERON:0000113
## 2 UBERON:0000473 testis UBERON:0000113
## 3 UBERON:0000473 testis UBERON:0000113
## 4 UBERON:0000473 testis UBERON:0000113
## 5 UBERON:0000473 testis UBERON:0000113
## 6 UBERON:0000473 testis UBERON:0000113
## Stage.name Sex Strain Read.count TPM FPKM
## 1 post-juvenile adult stage male C57BL/6 1240 11.779760 11.467125
## 2 post-juvenile adult stage male C57BL/6 0 0.000000 0.000000
## 3 post-juvenile adult stage male C57BL/6 585 10.046090 9.779467
## 4 post-juvenile adult stage male C57BL/6 71 2.325751 2.264026
## 5 post-juvenile adult stage male C57BL/6 718 7.448692 7.251004
## 6 post-juvenile adult stage male C57BL/6 782 27.757432 27.020750
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[7]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE30352 SRX081914 single ENSMUSG00000000001
## 2 GSE30352 SRX081914 single ENSMUSG00000000003
## 3 GSE30352 SRX081914 single ENSMUSG00000000028
## 4 GSE30352 SRX081914 single ENSMUSG00000000031
## 5 GSE30352 SRX081914 single ENSMUSG00000000037
## 6 GSE30352 SRX081914 single ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 2 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 3 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 4 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 5 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 6 UBERON:0000082 adult mammalian kidney UBERON:0000113
## Stage.name Sex Strain Read.count TPM FPKM
## 1 post-juvenile adult stage female C57BL/6 2870 39.095102 39.816500
## 2 post-juvenile adult stage female C57BL/6 0 0.000000 0.000000
## 3 post-juvenile adult stage female C57BL/6 51 1.343029 1.367811
## 4 post-juvenile adult stage female C57BL/6 15 0.682456 0.695049
## 5 post-juvenile adult stage female C57BL/6 11 0.253984 0.258671
## 6 post-juvenile adult stage female C57BL/6 2 0.077435 0.078864
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 present high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[8]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 GSE41637 SRX196275 paired ENSMUSG00000000001
## 2 GSE41637 SRX196275 paired ENSMUSG00000000003
## 3 GSE41637 SRX196275 paired ENSMUSG00000000028
## 4 GSE41637 SRX196275 paired ENSMUSG00000000031
## 5 GSE41637 SRX196275 paired ENSMUSG00000000037
## 6 GSE41637 SRX196275 paired ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 2 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 3 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 4 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 5 UBERON:0000082 adult mammalian kidney UBERON:0000113
## 6 UBERON:0000082 adult mammalian kidney UBERON:0000113
## Stage.name Sex Strain Read.count TPM FPKM
## 1 post-juvenile adult stage <NA> C57BL/6 8990 38.639422 31.498242
## 2 post-juvenile adult stage <NA> C57BL/6 0 0.000000 0.000000
## 3 post-juvenile adult stage <NA> C57BL/6 218 1.402498 1.143294
## 4 post-juvenile adult stage <NA> C57BL/6 9 0.043174 0.035195
## 5 post-juvenile adult stage <NA> C57BL/6 26 0.123947 0.101040
## 6 post-juvenile adult stage <NA> C57BL/6 788 8.513097 6.939742
## Detection.flag Detection.quality State.in.Bgee
## 1 present high quality Part of a call
## 2 absent high quality Result excluded, reason: pre-filtering
## 3 present high quality Part of a call
## 4 absent high quality Part of a call
## 5 present high quality Part of a call
## 6 present high quality Part of a call
##
## [[9]]
## Experiment.ID Library.ID Library.type Gene.ID
## 1 SRP022612 SRX277661 single ENSMUSG00000000001
## 2 SRP022612 SRX277661 single ENSMUSG00000000003
## 3 SRP022612 SRX277661 single ENSMUSG00000000028
## 4 SRP022612 SRX277661 single ENSMUSG00000000031
## 5 SRP022612 SRX277661 single ENSMUSG00000000037
## 6 SRP022612 SRX277661 single ENSMUSG00000000049
## Anatomical.entity.ID Anatomical.entity.name Stage.ID
## 1 UBERON:0000014 zone of skin MmusDv:0000062
## 2 UBERON:0000014 zone of skin MmusDv:0000062
## 3 UBERON:0000014 zone of skin MmusDv:0000062
## 4 UBERON:0000014 zone of skin MmusDv:0000062
## 5 UBERON:0000014 zone of skin MmusDv:0000062
## 6 UBERON:0000014 zone of skin MmusDv:0000062
## Stage.name Sex Strain Read.count TPM
## 1 2 month-old stage (mouse) male C57BL/6 5956 67.499246
## 2 2 month-old stage (mouse) male C57BL/6 0 0.000000
## 3 2 month-old stage (mouse) male C57BL/6 154 3.071474
## 4 2 month-old stage (mouse) male C57BL/6 40562 1047.442212
## 5 2 month-old stage (mouse) male C57BL/6 244 3.770360
## 6 2 month-old stage (mouse) male C57BL/6 3 0.218505
## FPKM Detection.flag Detection.quality
## 1 69.611952 present high quality
## 2 0.000000 absent high quality
## 3 3.167611 present high quality
## 4 1080.226848 present high quality
## 5 3.888371 present high quality
## 6 0.225344 present high quality
## State.in.Bgee
## 1 Part of a call
## 2 Result excluded, reason: pre-filtering
## 3 Part of a call
## 4 Part of a call
## 5 Part of a call
## 6 Part of a callThe result of the getData() function is, for each experiment, a data frame with the different samples listed in rows, one after the other. Each row is a gene and the expression levels are displayed as raw read counts or RPKMs. A detection flag indicates is the gene is significantly expressed above background level of expression.
Note 1: An additional column in the data frame including expression in the TPM unit will be available from Bgee release 14 (planned for the end of 2016).
Note 2: If microarray data are downloaded, rows correspond to probesets and log2 of expression intensities are available instead of read counts/RPKMs.
Alternatively, you can choose to download data from only one particular RNA-seq experiment from Bgee with the experimentId parameter:
##
## Downloading expression data for the experiment GSE30617 ...
##
## Saved expression data file in /tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Mus_musculus_Bgee_14_0 folder. Now unzip /tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Mus_musculus_Bgee_14_0/Mus_musculus_RNA-Seq_read_counts_TPM_FPKM_GSE30617.tsv.zip file...
##
Read 57.6% of 1718244 rows
Read 1718244 rows and 16 (of 16) columns from 0.284 GB file in 00:00:03
##
## Saving all data in .rds file...It is sometimes easier to work with data organized as a matrix, where rows represent genes or probesets and columns represent different samples. The formatData() function reformats the data into an ExpressionSet object including: * An expression data matrix, with genes or probesets as rows, and samples as columns (assayData slot). The stats argument allows to choose if the matrix should be filled with read counts, RPKMs (and soon TPMs) for RNA-seq data. For microarray data the matrix is filled with log2 expression intensities. * A data frame listing the samples and their anatomical structure and developmental stage annotation (phenoData slot) * For microarray data, the mapping from probesets to Ensembl genes (featureData slot)
The callType argument allows to retain only actively expressed genes or probesets, if set to “present” or “present high quality”. Genes or probesets that are absent in a given sample are given NA values.
# use only present calls and fill expression matric with RPKM values
gene.expression.mouse.rpkm <- formatData(bgee, data_bgee_mouse_gse30617, callType = "present", stats = "rpkm")##
## Extracting expression data matrix...
## Keeping only present genes.
##
## Extracting features information...
##
## Extracting samples information...## ExpressionSet (storageMode: lockedEnvironment)
## assayData: 47729 features, 36 samples
## element names: exprs
## protocolData: none
## phenoData
## sampleNames: ERX012344 ERX012345 ... ERX012379 (36 total)
## varLabels: Library.ID Anatomical.entity.ID ... Stage.name (5
## total)
## varMetadata: labelDescription
## featureData
## featureNames: ENSMUSG00000000001 ENSMUSG00000000003 ...
## ENSMUSG00000109578 (47729 total)
## fvarLabels: Gene.ID
## fvarMetadata: labelDescription
## experimentData: use 'experimentData(object)'
## Annotation:For some documentation on the TopAnat analysis, please refer to our publications, or to the web-tool page (http://bgee.org/?page=top_anat#/).
Similarly to the quantitative data download example above, the first step of a topAnat analysis is to built an object from the Bgee class. For this example, we will focus on zebrafish:
##
## NOTE: You did not specify any data type. The argument dataType will be set to c("rna_seq","affymetrix","est","in_situ") for the next steps.
##
## Querying Bgee to get release information...
##
## NOTE: the file describing Bgee species information for release 14_0 was found in the download directory /tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes. Data will not be redownloaded.
##
## API key built: 1a7bb93a14d2d550f3991414b52fe385384cf2e31833ed75cf43b32250dc915a7ba8f5d0fd07b4b79ce2469a5063eeadc17fddff08301180c8df6e131361491eNote : We are free to specify any data type of interest using the dataType argument among rna_seq, affymetrix, est or in_situ, or even a combination of data types. If nothing is specified, as in the above example, all data types available for the targeted species are used. This equivalent to specifying dataType=c("rna_seq","affymetrix","est","in_situ").
The loadTopAnatData() function loads a mapping from genes to anatomical structures based on calls of expression in anatomical structures. It also loads the structure of the anatomical ontology and the names of anatomical structures.
##
## Building URLs to retrieve organ relationships from Bgee.........
## URL successfully built (http://bgee.org/?page=r_package&action=get_anat_entity_relations&display_type=tsv&species_list=7955&attr_list=SOURCE_ID&attr_list=TARGET_ID&api_key=1a7bb93a14d2d550f3991414b52fe385384cf2e31833ed75cf43b32250dc915a7ba8f5d0fd07b4b79ce2469a5063eeadc17fddff08301180c8df6e131361491e&source=BgeeDB_R_package&source_version=2.5.0)
## Submitting URL to Bgee webservice (can be long)
## Got results from Bgee webservice. Files are written in "/tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Danio_rerio_Bgee_14_0"
##
## Building URLs to retrieve organ names from Bgee.................
## URL successfully built (http://bgee.org/?page=r_package&action=get_anat_entities&display_type=tsv&species_list=7955&attr_list=ID&attr_list=NAME&api_key=1a7bb93a14d2d550f3991414b52fe385384cf2e31833ed75cf43b32250dc915a7ba8f5d0fd07b4b79ce2469a5063eeadc17fddff08301180c8df6e131361491e&source=BgeeDB_R_package&source_version=2.5.0)
## Submitting URL to Bgee webservice (can be long)
## Got results from Bgee webservice. Files are written in "/tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Danio_rerio_Bgee_14_0"
##
## Building URLs to retrieve mapping of gene to organs from Bgee...
## URL successfully built (http://bgee.org/?page=r_package&action=get_expression_calls&display_type=tsv&species_list=7955&attr_list=GENE_ID&attr_list=ANAT_ENTITY_ID&api_key=1a7bb93a14d2d550f3991414b52fe385384cf2e31833ed75cf43b32250dc915a7ba8f5d0fd07b4b79ce2469a5063eeadc17fddff08301180c8df6e131361491e&source=BgeeDB_R_package&source_version=2.5.0&data_qual=SILVER)
## Submitting URL to Bgee webservice (can be long)
## Got results from Bgee webservice. Files are written in "/tmp/RtmpcJf98i/Rbuild9e564bf9984/BgeeDB/vignettes/Danio_rerio_Bgee_14_0"
##
## Parsing the results.............................................
##
## Adding BGEE:0 as unique root of all terms of the ontology.......
##
## Done.The strigency on the quality of expression calls can be changed with the confidence argument. Finally, if you are interested in expression data coming from a particular developmental stage or a group of stages, please specify the a Uberon stage Id in the stage argument.
## Loading only high-quality expression calls from affymetrix data made on embryonic samples only
## This is just given as an example, but is not run in this vignette because only few data are returned
bgee <- Bgee$new(species = "Danio_rerio", dataType="affymetrix")
myTopAnatData <- loadTopAnatData(bgee, stage="UBERON:0000068", confidence="high_quality")Note: As mentioned above, the downloaded data files are stored in a versioned folder that can be set with the pathToData argument when creating the Bgee class object (default is the working directory). If you query again Bgee with the exact same parameters, these cached files will be read instead of querying the web-service again. It is thus important, if you plan to reuse the same data for multiple parallel topAnat analyses, to plan to make use of these cached files instead of redownloading them for each analysis. The cached files also give the possibility to repeat analyses offline.
First we need to prepare a list of genes in the foreground and in the background. The input format is the same as the gene list required to build a topGOdata object in the topGO package: a vector with background genes as names, and 0 or 1 values depending if a gene is in the foreground or not. In this example we will look at genes, annotated with “spermatogenesis” or children terms in the Gene Ontology. We use the biomaRt package to retrieve this list of genes. We expect them to be enriched for expression in male tissues, notably the testes. The background list of genes is set to all genes annotated to at least one Gene Ontology term, allowing to account for biases in which types of genes are more likely to receive Gene Ontology annotation.
# source("https://bioconductor.org/biocLite.R")
# biocLite("biomaRt")
library(biomaRt)
ensembl <- useMart("ensembl")
ensembl <- useDataset("drerio_gene_ensembl", mart=ensembl)
# Foreground genes are those with GO annotation "spermatogenesis" or childrem terms
myGenes <- getBM(attributes= "ensembl_gene_id", filters=c("go_parent_term"), values=list(c("GO:0007283")), mart=ensembl)
# Background are all genes with GO annotation
universe <- getBM(attributes= "ensembl_gene_id", filters=c("with_go"), values=list(c(TRUE)), mart=ensembl)
# Prepare the gene list vector
geneList <- factor(as.integer(universe[,1] %in% myGenes[,1]))
names(geneList) <- universe[,1]
head(geneList)
summary(geneList == 1)
# Prepare the topGO object
myTopAnatObject <- topAnat(myTopAnatData, geneList)The above code using the biomaRt package is not executed in this vignette to prevent building issues of our package in case of biomaRt downtime. Instead we use a geneList object saved in the data/ folder that we built using pre-downloaded data.
##
## Checking topAnatData object.............
##
## Checking gene list......................
##
## WARNING: Some genes in your gene list have no expression data in Bgee, and will not be included in the analysis. 21801 genes in background will be kept.
##
## Building most specific Ontology terms... ( 1270 Ontology terms found. )
##
## Building DAG topology................... ( 2151 Ontology terms and 4102 relations. )
##
## Annotating nodes (Can be long).......... ( 21801 genes annotated to the Ontology terms. )Warning: This can be long, especially if the gene list is large, since the Uberon anatomical ontology is large and expression calls will be propagated through the whole ontology (e.g., expression in the forebrain will also be counted as expression in parent structures such as the brain, nervous system, etc). Consider running a script in batch mode if you have multiple analyses to do.
For this step, see the vignette of the topGO package for more details, as you have to directly use the tests implemented in the topGO package, as shown in this example:
##
## -- Classic Algorithm --
##
## the algorithm is scoring 298 nontrivial nodes
## parameters:
## test statistic: fisherYou can also choose one of the topGO decorrelation methods, for example the “weight” method, allowing to avoid redundant results induced by the structure of the ontology.
Warning: This can be long because of the size of the ontology. Consider running scripts in batch mode if you have multiple analyses to do.
The makeTable function allows to filter and format the test results, and calculate FDR values.
# Display results sigificant at a 10% FDR threshold
makeTable(myTopAnatData, myTopAnatObject, results, cutoff = 0.1)##
## Building the results table for the 5 significant terms at FDR threshold of 0.1...
## Ordering results by pValue column in increasing order...
## Done## organId organName annotated
## CL:0000039 CL:0000039 germ line cell 34
## UBERON:0000079 UBERON:0000079 male reproductive system 16563
## UBERON:0000473 UBERON:0000473 testis 16563
## UBERON:0003135 UBERON:0003135 male reproductive organ 16563
## UBERON:0003101 UBERON:0003101 male organism 16564
## significant expected foldEnrichment pValue FDR
## CL:0000039 3 0.06 50.000000 2.816424e-05 0.03523347
## UBERON:0000079 37 28.87 1.281607 3.768462e-04 0.09448179
## UBERON:0000473 37 28.87 1.281607 3.768462e-04 0.09448179
## UBERON:0003135 37 28.87 1.281607 3.768462e-04 0.09448179
## UBERON:0003101 37 28.87 1.281607 3.776251e-04 0.09448179At the time of building this vignette (April 2017), the significant terms were all related to male reproductive system (testes), which makes biological sense: there is an expression bias for testis of genes involved in spermatogenesis.
By default results are sorted by p-value, but this can be changed with the ordering parameter by specifying which column should be used to order the results (preceded by a “-” sign to indicate that ordering should be made in decreasing order). For example, it is often convenient to sort significant structures by decreasing enrichment fold, using ordering = -6. The full table of results can be obtained using cutoff = 1.
Warning: it is debated whether FDR correction is appropriate on enrichment test results, since tests on different terms of the ontologies are not independent. A nice discussion can be found in the vignette of the topGO package.