This package is a pipeline to identify the key gene regulators in a biological process, for example in cell differentiation and in cell development after stimulation. Given gene expression data and sample information, there are four major steps in this pipeline: (1) differential expression analysis; (2) regulator-target network inference; (3) enrichment analysis; and (4) regulators scoring and ranking.
In this tutorial, we are showing you how to perform RegEnrich analysis by starting with a quick example, followed by detailed explanation in each step and three case studies.
To illustrate how to use RegEnrich pipline, here we simply show the basic steps, assuming you have had all input data and the parameters ready.
object = RegenrichSet(expr = expressionMatrix, # expression data (matrix)
colData = sampleInfo, # sample information (data frame)
reg = regulators, # regulators
method = "limma", # differentila expression analysis method
design = designMatrix, # desing model matrix
contrast = contrast, # contrast
networkConstruction = "COEN", # network inference method
enrichTest = "FET") # enrichment analysis method# Differential expression analysis
object = regenrich_diffExpr(object)
# Regulator-target network inference
object = regenrich_network(object)
# Enrichment analysis
object = regenrich_enrich(object)
# Regulator scoring and ranking
object = regenrich_rankScore(object)
# Obtaining results
res = results_score(object)The code can be even simpler if pipe (%>%) is used.
# Perform 4 steps in RegEnrich analysis
object = regenrich_diffExpr(object) %>%
regenrich_network() %>%
regenrich_enrich() %>%
regenrich_rankScore()
res = results_score(object)RegEnrich package is programmed in a style of S4 object system. The most fundamental class
in RegEnrich is RegenrichSet, and majority functions are working with this class in the
following analysis steps. So the RegenrichSet object must be initialized prior to RegEnrich
analysis. The RegenrichSet object can be easily initialized by RegenrichSet function, which
require several input data along with other parameters.
There are three fundamental input data for RegEnrich pipline.
The first one is expression
data (expr), which is a table (matrix) with m rows (genes or proteins) and n columns (samples).
Here the expression data can be of genes, measured by microarray or
RNA sequencing, and can be of proteins, measured by mass spectrometry, etc.
Here we downloaded the gene expression data (RNA-sequencing data in FPKM format) that is from
(Bouquet et al. 2016) in GEO database
(GSE63085). And
only 52 samples were included in Lyme_GSE63085 dataset and can be loaded in the
following way.
Althouth here RNA-sequencing data is reprensented in FPKM (Fragments Per Kilobase of transcript per Million) format, we do recomment raw read count format. To further work on this FPKM data, we convert the FPKM data (plus 1) into logarithm to the base 2.
log2FPKM = log2(FPKM + 1)
print(log2FPKM[1:6, 1:5])
#> GSM1540487 GSM1540488 GSM1540489 GSM1540490 GSM1540491
#> A1BG 0.20255689 0.1035833 0.252087406 0.18977776 0.0000000
#> A1BG-AS1 0.24372879 0.3816776 0.256499850 0.67166657 0.0000000
#> A1CF 0.01262565 0.0000000 0.018093311 0.03190273 0.0000000
#> A2M 0.49206032 0.5511636 0.832497953 0.63190517 0.7838539
#> A2M-AS1 1.42709122 1.4919608 1.483632648 1.71557274 2.0862607
#> A2ML1 0.00000000 0.0000000 0.007600189 0.00000000 0.0000000The second input data is sample information (colData), which is also a table (data.frame),
showing which samples (rows) belonging to which groups or having what features (columns).
Here we use the sample information for the 52 samples in Lyme_GSE63085 dataset.
sampleInfo = Lyme_GSE63085$sampleInfo
head(sampleInfo)
#> geo_accession title source_name organism patientID week disease
#> GSM1540487 GSM1540487 01-20_V1 PBMCs Homo sapiens 01_20 0 Lyme disease
#> GSM1540488 GSM1540488 01-23_V1 PBMCs Homo sapiens 01_23 0 Lyme disease
#> GSM1540489 GSM1540489 01-24_V1 PBMCs Homo sapiens 01_24 0 Lyme disease
#> GSM1540490 GSM1540490 01-25_V1 PBMCs Homo sapiens 01_25 0 Lyme disease
#> GSM1540491 GSM1540491 01-26_V1 PBMCs Homo sapiens 01_26 0 Lyme disease
#> GSM1540492 GSM1540492 01-27_V1 PBMCs Homo sapiens 01_27 0 Lyme disease
#> description molecule
#> GSM1540487 acute Lyme pre-treatment (V1) total RNA
#> GSM1540488 acute Lyme pre-treatment (V1) total RNA
#> GSM1540489 acute Lyme pre-treatment (V1) total RNA
#> GSM1540490 acute Lyme pre-treatment (V1) total RNA
#> GSM1540491 acute Lyme pre-treatment (V1) total RNA
#> GSM1540492 acute Lyme pre-treatment (V1) total RNAThe third input is the regulators in the studied organisms. If the organism is human, RegEnrich by default provided transcrpiton factors and co-factors in humans as the regulators which are obtained from (Han et al. 2015; Marbach et al. 2016; and Liu et al. 2015).
data(TFs)
head(TFs)
#> TF TF_name
#> ENSG00000001167 ENSG00000001167 NFYA
#> ENSG00000004848 ENSG00000004848 ARX
#> ENSG00000005007 ENSG00000005007 UPF1
#> ENSG00000005073 ENSG00000005073 HOXA11
#> ENSG00000005075 ENSG00000005075 POLR2J
#> ENSG00000005102 ENSG00000005102 MEOX1You can define your own regulators in RegEnrich. For example, for the studies in other organisms such as mouse, yeast, drosophila and arabidopsis thaliana, you can use the transcription (co-)factors in the following links, and cite corresponding paper:
Mouse, Yeast, Drosophila, and Arabidopsis thaliana
But one thing to keep in mind, the type of names or IDs of regulators should be the same as those of genes in the expression data. For example, if ENSEMBL gene ID is used in the expression data, then the regulators should be represented by ENSEMBL gene ID as well.
In addition to previous 3 most fundamental input data, other parameters for RegEnrich analysis can be initialized here as well. These parameters include 3 groups.
First, parameters to perform differential expression analysis, such as method (differential
test method), minMeanExpr (the threshold to remove the lowly expressed gene), design
(design model matrix or formula), reduced (reduced model matrix or formula), contrast
(to extract the coefficients in the model), etc.
Here we consider the effect of different patients and time (week in sample information
table) on gene expression. To identify the differential genes related to time, we can simply use
LRT_LM method (likelihood retio test on two linear model) to perfrom differential expression
analysis. So the corresponding parameters are defined by:
Second, parameters to perform regulator-target network inference, such as networkConstruction
(network inference method), topNetPercent (what percentage of the top edges to retain), etc.
Here we use COEN method (weighted gene coexpression network) and other default parameters to
inference regulator-target network.
Third, parameters to perform enrichment analysis, such as enrichTest (enrichment method),
minSize (minimum number of targets of regulator), etc.
Here we use FET method (Fisher’s exact test) and other default parameters to perform
enrichment analysis.
The detailed explaination of these parameters can be found in the help page of RegenrichSet
function, which can be viewed by ?RegenrichSet. Unlike expression data and sample information
data, these parameters can be re-specified in the later steps of RegEnrich analysis.
To reduce the running time, we consider the first 2000 genes, and remove genes with mean log2FPKM <= 0.01.
object = RegenrichSet(expr = log2FPKM[1:2000, ],
colData = sampleInfo,
method = "LRT_LM",
minMeanExpr = 0.01,
design = ~ patientID + week,
reduced = ~ patientID,
networkConstruction = "COEN",
enrichTest = "FET")
print(object)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#> Differential expression analysis needs to be performed.In this step, the major goal is to obtain differential p-values and log2 fold changes of gene
expression between different conditions. There are couple of packages being developed to perform
differential expression analysis, such as
DESeq2,
edgeR,
limma,
DSS,
EBSeq, and
baySeq.
The full tutorials of these packages have been already provided as vignettes or other documentation in
these packages. Here, we provide a wraper function, regenrich_diffExpr, which allows you to choose
either “Wald_DESeq2”, “LRT_DESeq2”, “limma”, or “LRT_LM” to perform the differential expression analysis
on the RegenrichSet obejct.
Since RegenrichSet object is initialized with method = "LRT_LM",
regenrich_diffExpr function performs differential expression analysis using likelihood ratio test on
two linear models that are specified by design formula and reduced formula.
object = regenrich_diffExpr(object)
print(object)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#>
#> (1) 544 rows with differential p-value < 0.05
#>
#> Network inference needs to be performed, or a 'TopNetwork' object needs to be provided.
print(results_DEA(object))
#> DataFrame with 1720 rows and 3 columns
#> gene p logFC
#> <character> <numeric> <numeric>
#> A1BG A1BG 8.66920e-01 0
#> A1BG-AS1 A1BG-AS1 2.00275e-01 0
#> A1CF A1CF 6.55367e-01 0
#> A2M A2M 4.98931e-06 0
#> A2M-AS1 A2M-AS1 3.57559e-04 0
#> ... ... ... ...
#> C17orf103 C17orf103 0.80616307 0
#> C17orf104 C17orf104 0.00927184 0
#> C17orf105 C17orf105 0.71747166 0
#> C17orf107 C17orf107 0.96590666 0
#> C17orf47 C17orf47 0.72293696 0LRT_LM method is implemented for data with complicated experiment designs, in which it is less
meaningful to calculate log2 fold changes. In the current version of RegEnrich, calculating the
log2 fold change between conditions is not implemented in LRT_LM method. And the log2 fold
changes of all genes are set to 0 by default.
Here, parameters to perform differential expression analysis can be re-specified in
regenrich_diffExpr function. Below, we show an example of using limma to obtain differential
expressed genes, by changing parameters.
object2 = regenrich_diffExpr(object, method = "limma", coef = "week3")
#> When method is limma or Wald_DESeq2, the 'reduced' argument is not used.
#> The method is limma, a formula is provided to the design,
#> so the model matrix is generated using model.matrix(design, data = pData).
print(object2)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#>
#> (1) 510 rows with differential p-value < 0.05
#>
#> Network inference needs to be performed, or a 'TopNetwork' object needs to be provided.
print(results_DEA(object2))
#> DataFrame with 1720 rows and 3 columns
#> gene p logFC
#> <character> <numeric> <numeric>
#> A1BG A1BG 8.74264e-01 0.00409204
#> A1BG-AS1 A1BG-AS1 2.04362e-01 0.07395716
#> A1CF A1CF 8.38490e-01 -0.00224502
#> A2M A2M 8.23976e-05 0.27179291
#> A2M-AS1 A2M-AS1 1.10891e-03 0.30939387
#> ... ... ... ...
#> C17orf103 C17orf103 0.8071424 -0.01069455
#> C17orf104 C17orf104 0.0174435 -0.07552484
#> C17orf105 C17orf105 0.7972371 -0.00352343
#> C17orf107 C17orf107 0.9655112 -0.00305637
#> C17orf47 C17orf47 0.7496326 -0.00667404More detailed explaination of regenrich_diffExpr function can be accessed by ?regenrich_diffExpr.
RegEnrich provides two computational methods, COEN and GRN,
to infer regulator-target network based on expression data.
For COEN method, the weighted co-expression network is constructed using
WGCNA
package, and then the regulator-target network is the robust subnetwork
of weighted co-expression network, and the nodes and edges are removed if
they are not connected to any regulators.
With respect to GRN, it infers regulator-target network using random forest algorithm.
This method was initially described in
GENIE3 package,
and it is modified in RegEnrich to support parallel computing and to
control the model accuracy. Regulator-target network inferences using COEN and
GRN methods are shown bellow.
regenrich_network is the function to perform regulator-target network inference.
Since the networkConstruction = "COEN" parameter has been set during the RegenrichSet
initialization, so by default RegEnrich constructs a COEN network.
set.seed(123)
object = regenrich_network(object)
print(object)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#>
#> (1) 544 rows with differential p-value < 0.05
#>
#> (2) Top p% network info:
#> TopNetwork object (3661 edges, networkConstruction: 'COEN', percentage: 5%)
#> # A tibble: 3,661 × 3
#> set element weight
#> <chr> <chr> <dbl>
#> 1 AATF ABCB7 0.0213
#> 2 AATF ABCF2 0.0540
#> 3 AATF ABHD12 0.0233
#> 4 AATF ABI3 0.0342
#> 5 AATF ABTB2 0.0181
#> 6 AATF ACADM 0.0516
#> 7 ABT1 ACAP1 0.0297
#> 8 AATF ACAP2 0.0269
#> 9 AATF ACAT1 0.0231
#> 10 AATF ACBD5 0.0386
#> # … with 3,651 more rows
#> FET/GSEA enrichment needs to be performed.What happens under the hood in above codes is updating object@topNetP slot, which is a
Topnetwork class. RegEnrich provides a function to access this slot.
# TopNetwork class
print(results_topNet(object))
#> TopNetwork object (3661 edges, networkConstruction: 'COEN', percentage: 5%)
#> # A tibble: 3,661 × 3
#> set element weight
#> <chr> <chr> <dbl>
#> 1 AATF ABCB7 0.0213
#> 2 AATF ABCF2 0.0540
#> 3 AATF ABHD12 0.0233
#> 4 AATF ABI3 0.0342
#> 5 AATF ABTB2 0.0181
#> 6 AATF ACADM 0.0516
#> 7 ABT1 ACAP1 0.0297
#> 8 AATF ACAP2 0.0269
#> 9 AATF ACAT1 0.0231
#> 10 AATF ACBD5 0.0386
#> # … with 3,651 more rowsPlease note that since the oganism of GSE63085 dataset is Homo sapien, the regulators used in RegEnrich by default are obtained from (Han et al. 2015; Marbach et al. 2016; and Liu et al. 2015). And we are using gene names in the expression data, so the regulators here are also represented by gene names.
# All parameters are stored in object@paramsIn slot
head(slot(object, "paramsIn")$reg)
#> [1] "NFYA" "ARX" "UPF1" "HOXA11" "POLR2J" "MEOX1"Since network inference is generally very time-consuming, we suggest saving
the RegenrichSet object with the regulator-target network in case of
using it next time.
# Saving object to 'fileName.Rdata' file in '/folderName' directory
save(object, file = "/folderName/fileName.Rdata")A more detailed explaination can be found in the help pages of regenrich_network
(see ?regenrich_network) and TopNetwork-class (see ?"TopNetwork-class").
Alternatively, you can build a gene regulatory network (GRN) by setting
networkConstruction = "GRN" parameter in regenrich_network function.
To accelarate computing, you can set number of CPU cores and random seeds
using BPPARAM parameter.
Here you can control the accuracy of network inferance by minR which are computed
based on out-of-bag estimation in random forest. Please note that the lower minR
is set, the less edges and potential less regulators are retained.
### not run
library(BiocParallel)
# on non-Windows computers (use 2 workers)
bpparam = register(MulticoreParam(workers = 2, RNGseed = 1234))
# on Windows computers (use 2 workers)
bpparam = register(SnowParam(workers = 2, RNGseed = 1234))
object3 = regenrich_network(object, networkConstruction = "GRN",
BPPARAM = bpparam, minR = 0.3)
print(object3)
print(results_topNet(object3))
save(object3, file = "/folderName/fileName3.Rdata")It is also possible to provide a regulator-target network, which is obtained somewhere else.
For example, this network can be constructed based on the relation network of transcription
factors and their binding targets.
Here we assigned the constructed COEN regulator-target network (a Topnetwork object) in
object variable to object2 variable.
network_user = results_topNet(object)
print(class(network_user))
#> [1] "TopNetwork"
#> attr(,"package")
#> [1] "RegEnrich"
regenrich_network(object2) = network_user
print(object2)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#>
#> (1) 510 rows with differential p-value < 0.05
#>
#> (2) Top p% network info:
#> TopNetwork object (3661 edges, networkConstruction: 'new', percentage: 100%)
#> # A tibble: 3,661 × 3
#> set element weight
#> <chr> <chr> <dbl>
#> 1 AATF ABCB7 0.0213
#> 2 AATF ABCF2 0.0540
#> 3 AATF ABHD12 0.0233
#> 4 AATF ABI3 0.0342
#> 5 AATF ABTB2 0.0181
#> 6 AATF ACADM 0.0516
#> 7 ABT1 ACAP1 0.0297
#> 8 AATF ACAP2 0.0269
#> 9 AATF ACAT1 0.0231
#> 10 AATF ACBD5 0.0386
#> # … with 3,651 more rows
#> FET/GSEA enrichment needs to be performed.It is also fine to provide a 3-column table (data.frame object) of network edges, in which
the first column is regulators, the second column is targets, and the third column is edge
weight (reliability).
network_user = slot(results_topNet(object), "elementset")
print(class(network_user))
#> [1] "tbl_elementset" "tbl_elementset_base" "tbl_df" "tbl"
#> [5] "data.frame"
regenrich_network(object2) = as.data.frame(network_user)
print(object2)
#> RegenrichSet object
#> assayData: 1720 rows, 52 columns (filtered 280 rows by average expression <= 0.01)
#>
#> (1) 510 rows with differential p-value < 0.05
#>
#> (2) Top p% network info:
#> TopNetwork object (3661 edges, networkConstruction: 'new', percentage: 100%)
#> # A tibble: 3,661 × 3
#> set element weight
#> <chr> <chr> <dbl>
#> 1 AATF ABCB7 0.0213
#> 2 AATF ABCF2 0.0540
#> 3 AATF ABHD12 0.0233
#> 4 AATF ABI3 0.0342
#> 5 AATF ABTB2 0.0181
#> 6 AATF ACADM 0.0516
#> 7 ABT1 ACAP1 0.0297
#> 8 AATF ACAP2 0.0269
#> 9 AATF ACAT1 0.0231
#> 10 AATF ACBD5 0.0386
#> # … with 3,651 more rows
#> FET/GSEA enrichment needs to be performed.RegEnrich provides two methods to perform enrichment analysis, i.e. Fisher’s exact test (FET)
and gene set enrichment analysis (GSEA). Both methods are implemented in regenrich_enrich
function.
regenrich_enrich function updates object@resEnrich slot, which is a
Enrich class. RegEnrich provides results_enrich function to access this slot.
Since the enrichTest = "FET" parameter has been set during the RegenrichSet initialization,
so by default RegEnrich performs enrichment analysis using FET method.
object = regenrich_enrich(object)
print(results_enrich(object))
#> Enrich object (FET method, 65 regulators are used for enrichment,
#> 8 regulators pass the threshold)
#> # A tibble: 8 × 9
#> ID Description GeneRatio BgRatio pvalue p.adjust qvalue geneID Count
#> <chr> <chr> <chr> <chr> <dbl> <dbl> <dbl> <chr> <int>
#> 1 APBB1 APBB1 71/248 120/670 0.0000000427 0.00000181 0.00000152 APLP2/APOBEC2... 71
#> 2 ARNTL2 ARNTL2 21/248 23/670 0.0000000556 0.00000181 0.00000152 ARPC5/ARSB... 21
#> 3 ALYREF ALYREF 9/248 9/670 0.000119 0.00233 0.00196 ANKRD13A/ANLN... 9
#> 4 AES AES 32/248 52/670 0.000163 0.00233 0.00196 AGFG1/AGFG2... 32
#> 5 ARID3A ARID3A 30/248 48/670 0.000179 0.00233 0.00196 ARL11/ARL6IP5... 30
#> 6 ATF6 ATF6 35/248 60/670 0.000360 0.00390 0.00328 ATG4A/ATL3... 35
#> 7 BACH2 BACH2 22/248 34/670 0.000721 0.00669 0.00564 BANK1/BBS1... 22
#> 8 ARNT ARNT 37/248 72/670 0.00602 0.0489 0.0412 ARPC1B/ARPC4... 37
# enrich_FET = results_enrich(object)@allResult
enrich_FET = slot(results_enrich(object), "allResult")
head(enrich_FET[, 1:6])
#> # A tibble: 6 × 6
#> ID Description GeneRatio BgRatio pvalue p.adjust
#> <chr> <chr> <chr> <chr> <dbl> <dbl>
#> 1 APBB1 APBB1 71/248 120/670 0.0000000427 0.00000181
#> 2 ARNTL2 ARNTL2 21/248 23/670 0.0000000556 0.00000181
#> 3 ALYREF ALYREF 9/248 9/670 0.000119 0.00233
#> 4 AES AES 32/248 52/670 0.000163 0.00233
#> 5 ARID3A ARID3A 30/248 48/670 0.000179 0.00233
#> 6 ATF6 ATF6 35/248 60/670 0.000360 0.00390Since the enrichTest = "FET" parameter has been set during the RegenrichSet initialization,
but enrichTest = GSEA parameter can be re-specified in regenrich_enrich function to
perform enrichment analysis using GSEA method. Typically, GSEA is slower than FET method,
especially when the number of reg is large and the regulator-target network is also large.
Reducing the number of permutation (nperm, default is 10,000) can be a good trial to have a
look at preliminary results.
set.seed(123)
object2 = regenrich_enrich(object, enrichTest = "GSEA", nperm = 5000)
print(results_enrich(object))
#> Enrich object (FET method, 65 regulators are used for enrichment,
#> 8 regulators pass the threshold)
#> # A tibble: 8 × 9
#> ID Description GeneRatio BgRatio pvalue p.adjust qvalue geneID Count
#> <chr> <chr> <chr> <chr> <dbl> <dbl> <dbl> <chr> <int>
#> 1 APBB1 APBB1 71/248 120/670 0.0000000427 0.00000181 0.00000152 APLP2/APOBEC2... 71
#> 2 ARNTL2 ARNTL2 21/248 23/670 0.0000000556 0.00000181 0.00000152 ARPC5/ARSB... 21
#> 3 ALYREF ALYREF 9/248 9/670 0.000119 0.00233 0.00196 ANKRD13A/ANLN... 9
#> 4 AES AES 32/248 52/670 0.000163 0.00233 0.00196 AGFG1/AGFG2... 32
#> 5 ARID3A ARID3A 30/248 48/670 0.000179 0.00233 0.00196 ARL11/ARL6IP5... 30
#> 6 ATF6 ATF6 35/248 60/670 0.000360 0.00390 0.00328 ATG4A/ATL3... 35
#> 7 BACH2 BACH2 22/248 34/670 0.000721 0.00669 0.00564 BANK1/BBS1... 22
#> 8 ARNT ARNT 37/248 72/670 0.00602 0.0489 0.0412 ARPC1B/ARPC4... 37
# enrich_GSEA = results_enrich(object2)@allResult
enrich_GSEA = slot(results_enrich(object2), "allResult")
head(enrich_GSEA[, 1:6])
#> # A tibble: 6 × 6
#> regulator pval padj ES NES nMoreExtreme
#> <chr> <dbl> <dbl> <dbl> <dbl> <dbl>
#> 1 APBB1 0.000205 0.00554 0.567 2.01 0
#> 2 AES 0.000224 0.00554 0.591 1.92 0
#> 3 ARNTL2 0.000258 0.00554 0.833 2.34 0
#> 4 ALYREF 0.000316 0.00554 0.928 2.08 0
#> 5 ARID3A 0.00113 0.0159 0.576 1.84 4
#> 6 BHLHA15 0.00179 0.0190 0.900 1.74 4You can compare the order of enriched regulators obtained by FET and GSEA methods using
plotOrders function.
The RegEnrich score is a summarized information from both differential expression analysis and
regulator enrichment analysis for regulators. This step of RegEnrich analysis is done by
regenrich_rankScore function.
Above all, the differential expression analysis is perormed by LRT_LM method,
regulator-target network is infered by COEN method, and enrichment analysis is
performed by FET method, so the scores and ranking summarize the importance
of regulators by considering regulatory interactions in the studied biological process.
object = regenrich_rankScore(object)
results_score(object)
#> # A tibble: 65 × 5
#> reg negLogPDEA negLogPEnrich logFC score
#> * <chr> <dbl> <dbl> <dbl> <dbl>
#> 1 APBB1 4.70 7.37 0 1.71
#> 2 ARNTL2 3.95 7.25 0 1.58
#> 3 BACH2 5.03 3.14 0 1.19
#> 4 AEBP1 6.59 0.781 0 1.11
#> 5 AES 2.88 3.79 0 0.944
#> 6 ALYREF 2.75 3.92 0 0.943
#> 7 BRCA1 3.11 1.82 0 0.713
#> 8 ARID3A 1.19 3.75 0 0.680
#> 9 ATF6 1.34 3.44 0 0.662
#> 10 ARNT 2.00 2.22 0 0.597
#> # … with 55 more rowsThe expression of regulator and its targets can be viewed using following code.
Note that the previous analysis is a tutorial showing you how to perform basic RegEnrich analysis. As you known this analysis is based on only first 2000 genes, the real key regulators should be different from the previous results.
RegEnrich can work with different types of dataset, such as microarray data, RNAseq raw read count data, and mass spectrometriy proteomic data. The following section shows you 2 case studies of using RegEnrich to work with these 2 types of datasets.
This case study analyzes gene expression changes of primary human hepatocytes after 6 and 24 h treatment with interferon alpha (IFN-α). The gene expression was examined using single-channel Affymetrix Human Genome U133 Plus 2.0 Arrays. And the raw data and normalized data is available in GEO database under GSE31193 accession ID.
There are several ways to read the data.
You can download the normalized data file,
decompress it, and then read it using read.csv function.
Reading the raw data (.cel files) and then normalize it using other normalization method is also possible. As there is a simpler way to read data from GEO database, this method is not included in this vignette.
The simplist way to read the data is using GEOquery library. To use this library, you must
have this package installed.
if (!requireNamespace("GEOquery"))
BiocManager::install("GEOquery")
library(GEOquery)
eset <- getGEO(GEO = "GSE31193")[[1]]This dataset contains samples treated with IL28B, but here we are focusing on only control samples and samples after 6 and 24 h IFN-α treatment.
# Samples information
pd0 = pData(eset)
pd = pd0[, c("title", "time:ch1", "agent:ch1")]
colnames(pd) = c("title", "time", "group")
pd$time = factor(c(`n/a` = "0", `6` = "6", `24` = "24")[pd$time],
levels = c("0", "6", "24"))
pd$group = factor(pd$group, levels = c("none", "IFN", "IL28B"))
pData(eset) = pd
# Only samples without or after 6 and 24 h IFN-α treatment
eset_IFN = eset[, pd$group %in% c("none", "IFN")]
# Order the samples based on time of treatment
pData(eset_IFN) = pData(eset_IFN)[order(pData(eset_IFN)$time),]
# Rename samples
colnames(eset_IFN) = pData(eset_IFN)$title
# Probes information
probeGene = fData(eset_IFN)[, "Gene Symbol", drop = FALSE]Here to simplify the analysis, if there are multiple probes matching a gene, we use only one probe with higher average expression value to represent this gene.
probeGene$meanExpr = rowMeans(exprs(eset_IFN))
probeGene = probeGene[order(probeGene$meanExpr, decreasing = TRUE),]
# Keep a single probe for a gene, and remove the probe matching no gene.
probeGene_noDu = probeGene[!duplicated(probeGene$`Gene Symbol`), ][-1,]
data = eset_IFN[rownames(probeGene_noDu), ]
rownames(data) = probeGene_noDu$`Gene Symbol`Because the speed of network infernece is highly influenced by the number of genes, to quickly illustrate how to use RegEnrich, here we randomly take only 5,000 genes for analysis. If you would like to see the real result in the analysis, then the following data subsetting step should be discarded.
Here we would like to know which regulators play key roles in primary human hepatocytes after 24 h treatment with IFN-α.
expressionMatrix = exprs(data) # expression data
# rownames(expressionMatrix) = probeGene_noDu$`Gene Symbol`
sampleInfo = pData(data) # sample information
design = ~time
contrast = c(0, 0, 1) # to extract the coefficient "time24"
data(TFs)
# Initializing a RegenrichSet object
object = RegenrichSet(expr = expressionMatrix, # expression data (matrix)
colData = sampleInfo, # sample information (data frame)
reg = unique(TFs$TF_name), # regulators
method = "limma", # differentila expression analysis method
design = design, # desing fomula
contrast = contrast, # contrast
networkConstruction = "COEN", # network inference method
enrichTest = "FET") # enrichment analysis method
# Perform RegEnrich analysis
set.seed(123)
# This procedure takes quite a bit of time.
object = regenrich_diffExpr(object) %>%
regenrich_network() %>%
regenrich_enrich() %>%
regenrich_rankScore()res = results_score(object)
res
#> # A tibble: 133 × 5
#> reg negLogPDEA negLogPEnrich logFC score
#> * <chr> <dbl> <dbl> <dbl> <dbl>
#> 1 NMI 8.48 63.5 1.56 1.79
#> 2 IRF7 7.49 59.4 2.30 1.63
#> 3 ETV6 5.91 53.4 1.26 1.39
#> 4 ETV7 10.7 4.20 3.92 1.07
#> 5 NOTCH2 2.31 42.3 -0.261 0.881
#> 6 DCP1A 5.51 16.1 1.06 0.768
#> 7 EIF2AK2 6.86 6.73 1.24 0.747
#> 8 CIITA 6.26 9.27 1.56 0.731
#> 9 IRF9 5.99 9.27 1.53 0.705
#> 10 ETS2 2.14 27.4 0.232 0.631
#> # … with 123 more rowsHere we show how to apply RegEnrich on the RNAseq data by analyzing Kang et al’s monocyte-macrophage-IFN stimulation dataset ( GSE130567). There are multiple experiment conditions in this study. But here we would like to focus on partial samples in which monocytes were cultured with 10 ng/ml human macrophage colonystimulating factor (M-CSF) in the presence (IFN-γ-primed macrophages) or absence (resting macrophages) of 100 U/ml human IFN-γ for 48 h. RNA were extracted and reverse transcripted followed by sequencing (50 bp, paired-end) using Illumina HiSeq 4000. Sequenced reads were mapped to reference human genome (hg19 assembly) using STAR aligner with default parameters. We will use the raw HT-seq counts for the RegEnrich analysis.
Since the sample information and raw read counts data are archived seperately in GEO database.
First, we can read the sample information using GEOquery package.
library(GEOquery)
eset <- getGEO(GEO = "GSE130567")[[1]]
pdata = pData(eset)[, c("title", "geo_accession", "cultured in:ch1", "treatment:ch1")]
colnames(pdata) = c("title", "accession", "cultured", "treatment")
pData(eset) = pdata
# Only samples cultured with M-CSF in the presence or absence of IFN-γ
eset = eset[, pdata$treatment %in% c("NT", "IFNG-3h") & pdata$cultured == "M-CSF"]
# Sample information
sampleInfo = pData(eset)
rownames(sampleInfo) = paste0(rep(c("Resting", "IFNG"), each = 3), 1:3)
sampleInfo$treatment = factor(rep(c("Resting", "IFNG"), each = 3),
levels = c("Resting", "IFNG"))Second, download read count file and decompose into a temporary folder.
tmpFolder = tempdir()
tmpFile = tempfile(pattern = "GSE130567_", tmpdir = tmpFolder, fileext = ".tar")
download.file("https://www.ncbi.nlm.nih.gov/geo/download/?acc=GSE130567&format=file",
destfile = tmpFile, mode = "wb")
untar(tmpFile, exdir = tmpFolder)
files = untar(tmpFile, list = TRUE)
filesFull = file.path(tmpFolder, files)Then read the raw read counts in these files.
dat = list()
for (file in filesFull){
accID = gsub(".*(GSM\\d{7}).*", "\\1", file)
if(accID %in% sampleInfo$accession){
zz = gzfile(file, "rt")
zzdata = read.csv(zz, header = FALSE, sep = "\t", skip = 4, row.names = 1)
close(zz)
zzdata = zzdata[,1, drop = FALSE] # Extract the first numeric column
colnames(zzdata) = accID
dat = c(dat, list(zzdata))
}
}
edata = do.call(cbind, dat)
edata = edata[grep(".*[0-9]+$", rownames(edata)),] # remove PAR locus genes
rownames(edata) = substr(rownames(edata), 1, 15)
colnames(edata) = rownames(sampleInfo)
# Retain genes with average read counts higher than 1
edata = edata[rowMeans(edata) > 1,]Similar to the case 1, here we randomly take only 5,000 genes to quickly illustrate how to use RegEnrich, but to see the real result from the analysis, you should neglect the following step.
expressionMatrix = as.matrix(edata) # expression data
design = ~ treatment
reduced = ~ 1
data(TFs)
# Initializing a RegenrichSet object
object = RegenrichSet(expr = expressionMatrix, # expression data (matrix)
colData = sampleInfo, # sample information (data frame)
reg = unique(TFs$TF), # regulators
method = "LRT_DESeq2", # differentila expression analysis method
design = design, # desing fomula
reduced = reduced, # reduced
networkConstruction = "COEN", # network inference method
enrichTest = "FET") # enrichment analysis method
# Perform RegEnrich analysis
set.seed(123)
# This procedure takes quite a bit of time.
object = regenrich_diffExpr(object) %>%
regenrich_network() %>%
regenrich_enrich() %>%
regenrich_rankScore()res = results_score(object)
res$name = TFs[res$reg, "TF_name"]
res
#> # A tibble: 91 × 6
#> reg negLogPDEA negLogPEnrich logFC score name
#> * <chr> <dbl> <dbl> <dbl> <dbl> <chr>
#> 1 ENSG00000028277 0.608 49.0 -0.359 1.10 POU2F2
#> 2 ENSG00000153071 6.16 0.786 -1.37 1.02 DAB2
#> 3 ENSG00000125520 6.04 0.602 -1.52 0.992 SLC2A4RG
#> 4 ENSG00000181090 2.34 27.6 -0.878 0.944 EHMT1
#> 5 ENSG00000055332 4.28 0.434 -1.05 0.703 EIF2AK2
#> 6 ENSG00000153234 3.84 0.590 -1.62 0.634 NR4A2
#> 7 ENSG00000155229 1.50 18.9 -0.692 0.629 MMS19
#> 8 ENSG00000159692 1.10 13.6 -0.468 0.456 CTBP1
#> 9 ENSG00000116560 2.67 0.276 -0.871 0.438 SFPQ
#> 10 ENSG00000100105 2.69 0.0427 -0.953 0.437 PATZ1
#> # … with 81 more rowssessionInfo()
#> R version 4.2.0 RC (2022-04-19 r82224)
#> Platform: x86_64-pc-linux-gnu (64-bit)
#> Running under: Ubuntu 20.04.4 LTS
#>
#> Matrix products: default
#> BLAS: /home/biocbuild/bbs-3.15-bioc/R/lib/libRblas.so
#> LAPACK: /home/biocbuild/bbs-3.15-bioc/R/lib/libRlapack.so
#>
#> locale:
#> [1] LC_CTYPE=en_US.UTF-8 LC_NUMERIC=C LC_TIME=en_GB
#> [4] LC_COLLATE=C LC_MONETARY=en_US.UTF-8 LC_MESSAGES=en_US.UTF-8
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#> [10] LC_TELEPHONE=C LC_MEASUREMENT=en_US.UTF-8 LC_IDENTIFICATION=C
#>
#> attached base packages:
#> [1] stats4 stats graphics grDevices utils datasets methods base
#>
#> other attached packages:
#> [1] GEOquery_2.64.0 RegEnrich_1.6.0 SummarizedExperiment_1.26.0
#> [4] Biobase_2.56.0 GenomicRanges_1.48.0 GenomeInfoDb_1.32.0
#> [7] IRanges_2.30.0 MatrixGenerics_1.8.0 matrixStats_0.62.0
#> [10] BiocSet_1.10.0 tibble_3.1.6 dplyr_1.0.8
#> [13] S4Vectors_0.34.0 BiocGenerics_0.42.0 BiocStyle_2.24.0
#>
#> loaded via a namespace (and not attached):
#> [1] fgsea_1.22.0 colorspace_2.0-3 ellipsis_0.3.2 dynamicTreeCut_1.63-1
#> [5] htmlTable_2.4.0 qvalue_2.28.0 XVector_0.36.0 base64enc_0.1-3
#> [9] rstudioapi_0.13 bit64_4.0.5 AnnotationDbi_1.58.0 fansi_1.0.3
#> [13] xml2_1.3.3 R.methodsS3_1.8.1 codetools_0.2-18 splines_4.2.0
#> [17] doParallel_1.0.17 impute_1.70.0 cachem_1.0.6 GOSemSim_2.22.0
#> [21] geneplotter_1.74.0 knitr_1.38 Formula_1.2-4 jsonlite_1.8.0
#> [25] annotate_1.74.0 WGCNA_1.71 cluster_2.1.3 GO.db_3.15.0
#> [29] R.oo_1.24.0 png_0.1-7 readr_2.1.2 BiocManager_1.30.17
#> [33] compiler_4.2.0 httr_1.4.2 backports_1.4.1 assertthat_0.2.1
#> [37] Matrix_1.4-1 fastmap_1.1.0 ontologyIndex_2.7 limma_3.52.0
#> [41] cli_3.3.0 htmltools_0.5.2 tools_4.2.0 gtable_0.3.0
#> [45] glue_1.6.2 GenomeInfoDbData_1.2.8 reshape2_1.4.4 DO.db_2.9
#> [49] fastmatch_1.1-3 Rcpp_1.0.8.3 jquerylib_0.1.4 vctrs_0.4.1
#> [53] Biostrings_2.64.0 preprocessCore_1.58.0 iterators_1.0.14 xfun_0.30
#> [57] fastcluster_1.2.3 stringr_1.4.0 lifecycle_1.0.1 XML_3.99-0.9
#> [61] DOSE_3.22.0 zlibbioc_1.42.0 scales_1.2.0 hms_1.1.1
#> [65] parallel_4.2.0 RColorBrewer_1.1-3 curl_4.3.2 yaml_2.3.5
#> [69] memoise_2.0.1 gridExtra_2.3 ggplot2_3.3.5 sass_0.4.1
#> [73] rpart_4.1.16 latticeExtra_0.6-29 stringi_1.7.6 RSQLite_2.2.12
#> [77] genefilter_1.78.0 BiocIO_1.6.0 randomForest_4.7-1 foreach_1.5.2
#> [81] checkmate_2.1.0 BiocParallel_1.30.0 rlang_1.0.2 pkgconfig_2.0.3
#> [85] bitops_1.0-7 evaluate_0.15 lattice_0.20-45 purrr_0.3.4
#> [89] htmlwidgets_1.5.4 bit_4.0.4 tidyselect_1.1.2 plyr_1.8.7
#> [93] magrittr_2.0.3 bookdown_0.26 DESeq2_1.36.0 R6_2.5.1
#> [97] generics_0.1.2 Hmisc_4.7-0 DelayedArray_0.22.0 DBI_1.1.2
#> [101] pillar_1.7.0 foreign_0.8-82 survival_3.3-1 KEGGREST_1.36.0
#> [105] RCurl_1.98-1.6 nnet_7.3-17 crayon_1.5.1 utf8_1.2.2
#> [109] tzdb_0.3.0 rmarkdown_2.14 jpeg_0.1-9 locfit_1.5-9.5
#> [113] grid_4.2.0 data.table_1.14.2 blob_1.2.3 digest_0.6.29
#> [117] xtable_1.8-4 tidyr_1.2.0 R.utils_2.11.0 munsell_0.5.0
#> [121] bslib_0.3.1Bouquet, Jerome, Mark J Soloski, Andrea Swei, Chris Cheadle, Scot Federman, Jean-Noel Billaud, Alison W Rebman, et al. 2016. “Longitudinal Transcriptome Analysis Reveals a Sustained Differential Gene Expression Signature in Patients Treated for Acute Lyme Disease.” MBio 7 (1): e00100–16.
Han, Heonjong, Hongseok Shim, Donghyun Shin, Jung Eun Shim, Yunhee Ko, Junha Shin, Hanhae Kim, et al. 2015. “TRRUST: A Reference Database of Human Transcriptional Regulatory Interactions.” Scientific Reports 5: 11432.
Liu, Zhi-Ping, Canglin Wu, Hongyu Miao, and Hulin Wu. 2015. “RegNetwork: An Integrated Database of Transcriptional and Post-Transcriptional Regulatory Networks in Human and Mouse.” Database 2015.
Marbach, Daniel, David Lamparter, Gerald Quon, Manolis Kellis, Zoltán Kutalik, and Sven Bergmann. 2016. “Tissue-Specific Regulatory Circuits Reveal Variable Modular Perturbations Across Complex Diseases.” Nature Methods 13 (4): 366.