| Title: | An R package for the analysis and result reporting of RNA-Seq data by combining multiple statistical algorithms |
|---|---|
| Description: | Provides an interface to several normalization and statistical testing packages for RNA-Seq gene expression data. Additionally, it creates several diagnostic plots, performs meta-analysis by combinining the results of several statistical tests and reports the results in an interactive way. |
| Authors: | Panagiotis Moulos [aut, cre] |
| Maintainer: | Panagiotis Moulos <[email protected]> |
| License: | GPL (>= 3) |
| Version: | 1.17.0 |
| Built: | 2024-07-12 05:31:00 UTC |
| Source: | https://github.com/bioc/metaseqR2 |
This function creates a local annotation database to be used with metaseqr2 so as to avoid long time on the fly annotation downloads and formatting.
buildAnnotationDatabase(organisms, sources, db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), forceDownload = TRUE, rc = NULL)buildAnnotationDatabase(organisms, sources, db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), forceDownload = TRUE, rc = NULL)
organisms |
a list of organisms and versions
for which to download and build annotations. Check
the main |
sources |
a character vector of public sources
from which to download and build annotations. Check
the main |
db |
a valid path (accessible at least by the
current user) where the annotation database will be
set up. It defaults to
|
forceDownload |
by default,
|
rc |
fraction (0-1) of cores to use in a multicore
system. It defaults to |
Regarding the organisms argument, it is a list
with specific format which instructs
buildAnnotationDatabase on which organisms and
versions to download from the respective sources. Such
a list may have the format:
organisms=list(hg19=75, mm9=67, mm10=96:97)
This is explained as follows:
A database comprising the human genome versions
hg19 and the mouse genome versions
mm9, mm10 will be constructed.
If "ensembl" is in sources,
version 75 is downloaded for hg19 and versions
67, 96, 97 for mm9, mm10.
If "ucsc" or "refseq" are in
sources, the latest versions are downloaded
and marked by the download date. As UCSC and RefSeq
versions are not accessible in the same way as
Ensembl, this procedure cannot always be replicated.
organisms can also be a character vector with organism
names/versions (e.g. organisms = c("mm10","hg19")),
then the latest versions are downloaded in the case of
Ensembl.
Regarding db, this controls the location of the
installation database. If the default is used, then there is
no need to provide the local database path to any function
that uses the database (e.g. the main metaseqr2).
Otherwise, the user will either have to provide this each
time, or the annotation will have to be downloaded and used
on-the-fly.
The function does not return anything. Only the SQLite database is created or updated.
Panagiotis Moulos
# Build a test database with one genome myDb <- file.path(tempdir(),"testann.sqlite") organisms <- list(mm10=75) sources <- "ensembl" # If the example is not running in a multicore system, rc is ignored #buildAnnotationDatabase(organisms,sources,db=myDb,rc=0.5) # A more complete case, don't run as example # Since we are using Ensembl, we can also ask for a version #organisms <- list( # mm9=67, # mm10=96:97, # hg19=75, # hg38=96:97 #) #sources <- c("ensembl", "refseq") ## Build on the default location (depending on package location, it may ## require root/sudo) #buildAnnotationDatabase(organisms,sources) ## Build on an alternative location #myDb <- file.path(path.expand("~"),"my_ann.sqlite") #buildAnnotationDatabase(organisms,sources,db=myDb)# Build a test database with one genome myDb <- file.path(tempdir(),"testann.sqlite") organisms <- list(mm10=75) sources <- "ensembl" # If the example is not running in a multicore system, rc is ignored #buildAnnotationDatabase(organisms,sources,db=myDb,rc=0.5) # A more complete case, don't run as example # Since we are using Ensembl, we can also ask for a version #organisms <- list( # mm9=67, # mm10=96:97, # hg19=75, # hg38=96:97 #) #sources <- c("ensembl", "refseq") ## Build on the default location (depending on package location, it may ## require root/sudo) #buildAnnotationDatabase(organisms,sources) ## Build on an alternative location #myDb <- file.path(path.expand("~"),"my_ann.sqlite") #buildAnnotationDatabase(organisms,sources,db=myDb)
This function imports a GTF file with some custom annotation to the metaseqR2 annotation database.
buildCustomAnnotation(gtfFile, metadata, db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), rewrite=TRUE)buildCustomAnnotation(gtfFile, metadata, db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), rewrite=TRUE)
gtfFile |
a GTF file containing the gene structure of the organism to be imported. |
metadata |
a list with additional information about the annotation to be imported. See Details. |
db |
a valid path (accessible at least by the
current user) where the annotation database will be
set up. It defaults to
|
rewrite |
if custom annotation found, rwrite?
(default |
Regarding the metadata argument, it is a list
with specific format which instructs
buildCustomAnnotation on importing the custom
annotation. Such a list may has the following members:
organism a name of the organism which is
imported (e.g. "my_mm9"). This is the only
mandatory member.
source a name of the source for this
custom annotation (e.g. "my_mouse_db"). If
not given or NULL, the word "inhouse"
is used.
version a string denoting the version.
If not given or NULL, current date is used.
chromInfo it can be one of the following:
a tab-delimited file with two columns, the first being the chromosome/sequence names and the second being the chromosome/sequence lengths.
a BAM file to read the header from and obtain the required information
a data.frame with one column
with chromosome lengths and chromosome names as
rownames.
See the examples below for a metadata example.
Regarding db, this controls the location of the
installation database. If the default is used, then there is
no need to provide the local database path to any function
that uses the database (e.g. the main metaseqr2).
Otherwise, the user will either have to provide this each
time, or the annotation will have to be downloaded and used
on-the-fly.
The function does not return anything. Only the SQLite database is created or updated.
Panagiotis Moulos
# Dummy database as example customDir <- file.path(tempdir(),"test_custom") dir.create(customDir) myDb <- file.path(customDir,"testann.sqlite") chromInfo <- data.frame(length=c(1000L,2000L,1500L), row.names=c("A","B","C")) # Build with the metadata list filled (you can also provide a version) if (.Platform$OS.type == "unix") { buildCustomAnnotation( gtfFile=file.path(system.file(package="metaseqR2"), "dummy.gtf"), metadata=list( organism="dummy", source="dummy_db", version=1, chromInfo=chromInfo ), db=myDb ) # Try to retrieve some data myGenes <- loadAnnotation(genome="dummy",refdb="dummy_db", level="gene",type="gene",db=myDb) myGenes } ## Real data! ## Setup a temporary directory to download files etc. #customDir <- file.path(tempdir(),"test_custom") #dir.create(customDir) #myDb <- file.path(customDir,"testann.sqlite") ## Gene annotation dump from Ensembl #download.file(paste0("ftp://ftp.ensembl.org/pub/release-98/gtf/", # "dasypus_novemcinctus/Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # file.path(customDir,"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz")) ## Chromosome information will be provided from the following BAM file ## available from Ensembl #bamForInfo <- paste0("ftp://ftp.ensembl.org/pub/release-98/bamcov/", # "dasypus_novemcinctus/genebuild/Dasnov3.broad.Ascending_Colon_5.1.bam") ## Build with the metadata list filled (you can also provide a version) #buildCustomAnnotation( # gtfFile=file.path(customDir,"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # metadata=list( # organism="dasNov3_test", # source="ensembl_test", # chromInfo=bamForInfo # ), # db=myDb #) ## Try to retrieve some data #dasGenes <- loadAnnotation(genome="dasNov3_test",refdb="ensembl_test", # level="gene",type="gene",db=myDb) #dasGenes# Dummy database as example customDir <- file.path(tempdir(),"test_custom") dir.create(customDir) myDb <- file.path(customDir,"testann.sqlite") chromInfo <- data.frame(length=c(1000L,2000L,1500L), row.names=c("A","B","C")) # Build with the metadata list filled (you can also provide a version) if (.Platform$OS.type == "unix") { buildCustomAnnotation( gtfFile=file.path(system.file(package="metaseqR2"), "dummy.gtf"), metadata=list( organism="dummy", source="dummy_db", version=1, chromInfo=chromInfo ), db=myDb ) # Try to retrieve some data myGenes <- loadAnnotation(genome="dummy",refdb="dummy_db", level="gene",type="gene",db=myDb) myGenes } ## Real data! ## Setup a temporary directory to download files etc. #customDir <- file.path(tempdir(),"test_custom") #dir.create(customDir) #myDb <- file.path(customDir,"testann.sqlite") ## Gene annotation dump from Ensembl #download.file(paste0("ftp://ftp.ensembl.org/pub/release-98/gtf/", # "dasypus_novemcinctus/Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # file.path(customDir,"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz")) ## Chromosome information will be provided from the following BAM file ## available from Ensembl #bamForInfo <- paste0("ftp://ftp.ensembl.org/pub/release-98/bamcov/", # "dasypus_novemcinctus/genebuild/Dasnov3.broad.Ascending_Colon_5.1.bam") ## Build with the metadata list filled (you can also provide a version) #buildCustomAnnotation( # gtfFile=file.path(customDir,"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # metadata=list( # organism="dasNov3_test", # source="ensembl_test", # chromInfo=bamForInfo # ), # db=myDb #) ## Try to retrieve some data #dasGenes <- loadAnnotation(genome="dasNov3_test",refdb="ensembl_test", # level="gene",type="gene",db=myDb) #dasGenes
This function combines p-values from the
various statistical tests supported by
metaseqR2 using the Bonferroni's method (see
reference in the main metaseqr2
help page or in the vignette).
combineBonferroni(p, zerofix = NULL)combineBonferroni(p, zerofix = NULL)
p |
a vector of p-values for each statistical tests). |
zerofix |
|
The argument zerofix is used to correct for
the case of a p-value which is equal to 0 as a result
of internal numerical and approximation procedures.
When NULL, random numbers greater than 0 and
less than or equal to 0.5 are used to multiply the
offending p-values with the lowest provided non-zero
p-value, maintaining thus a virtual order of
significance, avoiding having the same p-values for
two tests and assuming that all zero p-values represent
extreme statistical significance. When a numeric
between 0 and 1, this number is used for the above
multiplication instead.
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineBonferroni(p)p <- matrix(runif(300),100,3) pc <- combineBonferroni(p)
This function combines p-values from the various statistical tests supported by metaseqR using p-value weights.
combineHarmonic(p, w, zerofix = NULL)combineHarmonic(p, w, zerofix = NULL)
p |
a p-value matrix (rows are genes, columns are statistical tests). |
w |
a weights vector, must sum to 1. |
zerofix |
|
The argument zerofix is used to correct for
the case of a p-value which is equal to 0 as a result
of internal numerical and approximation procedures.
When NULL, random numbers greater than 0 and
less than or equal to 0.5 are used to multiply the
offending p-values with the lowest provided non-zero
p-value, maintaining thus a virtual order of
significance, avoiding having the same p-values for
two tests and assuming that all zero p-values represent
extreme statistical significance. When a numeric
between 0 and 1, this number is used for the above
multiplication instead.
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineHarmonic(p,w=c(0.2,0.5,0.3))p <- matrix(runif(300),100,3) pc <- combineHarmonic(p,w=c(0.2,0.5,0.3))
This function combines p-values from the various statistical tests supported by metaseqR by taking the maximum p-value.
combineMaxp(p)combineMaxp(p)
p |
a p-value matrix (rows are genes, columns are statistical tests). |
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineMaxp(p)p <- matrix(runif(300),100,3) pc <- combineMaxp(p)
This function combines p-values from the various statistical tests supported by metaseqR by taking the minimum p-value.
combineMinp(p)combineMinp(p)
p |
a p-value matrix (rows are genes, columns are statistical tests). |
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineMinp(p)p <- matrix(runif(300),100,3) pc <- combineMinp(p)
This function combines p-values from the
various statistical tests supported by
metaseqR using the Simes' method (see
reference in the main metaseqr2
help page or in the vignette).
combineSimes(p, zerofix = NULL)combineSimes(p, zerofix = NULL)
p |
a p-value matrix (rows are genes, columns are statistical tests). |
zerofix |
|
The argument zerofix is used to correct for
the case of a p-value which is equal to 0 as a result
of internal numerical and approximation procedures.
When NULL, random numbers greater than 0 and
less than or equal to 0.5 are used to multiply the
offending p-values with the lowest provided non-zero
p-value, maintaining thus a virtual order of
significance, avoiding having the same p-values for
two tests and assuming that all zero p-values represent
extreme statistical significance. When a numeric
between 0 and 1, this number is used for the above
multiplication instead.
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineSimes(p)p <- matrix(runif(300),100,3) pc <- combineSimes(p)
This function combines p-values from the various statistical tests supported by metaseqR using p-value weights.
combineWeight(p, w, zerofix = NULL)combineWeight(p, w, zerofix = NULL)
p |
a p-value matrix (rows are genes, columns are statistical tests). |
w |
a weights vector, must sum to 1. |
zerofix |
|
The argument zerofix is used to correct for
the case of a p-value which is equal to 0 as a result
of internal numerical and approximation procedures.
When NULL, random numbers greater than 0 and
less than or equal to 0.5 are used to multiply the
offending p-values with the lowest provided non-zero
p-value, maintaining thus a virtual order of
significance, avoiding having the same p-values for
two tests and assuming that all zero p-values represent
extreme statistical significance. When a numeric
between 0 and 1, this number is used for the above
multiplication instead.
A vector of combined p-values.
Panagiotis Moulos
p <- matrix(runif(300),100,3) pc <- combineWeight(p,w=c(0.2,0.5,0.3))p <- matrix(runif(300),100,3) pc <- combineWeight(p,w=c(0.2,0.5,0.3))
This function creates bigWig files to be used for exploring RNA signal in genome browsers. When strands are separated, a UCSC genome browser trackhub is created to group tracks for the same sample. A link to the created data is returned.
createSignalTracks(targets, org, urlBase = NULL, stranded = FALSE, normTo = 1e+9, exportPath = ".", hubInfo = list(name = "MyHub", shortLabel = "My hub", longLabel = "My hub", email = "[email protected]"), fasta = NULL, gtf = NULL, forceHub = FALSE, overwrite = FALSE, rc = NULL)createSignalTracks(targets, org, urlBase = NULL, stranded = FALSE, normTo = 1e+9, exportPath = ".", hubInfo = list(name = "MyHub", shortLabel = "My hub", longLabel = "My hub", email = "[email protected]"), fasta = NULL, gtf = NULL, forceHub = FALSE, overwrite = FALSE, rc = NULL)
targets |
a tab-delimited file with the experimental
description or the output of |
org |
See the |
urlBase |
a valid URL which is prepended to the created bigWig files. |
stranded |
Separate + and - strands and create separate bigWig files. |
normTo |
the total sum of signal to be used as the
normalization target. See also the |
exportPath |
path to export tracks. |
hubInfo |
information regarding the track hub created
when |
overwrite |
overwrite tracks if they exist? Defaults to
|
fasta |
reference genome in FASTA format for the case of analyzing a custom, non-directly supported organism. It will be converted to the .2bit format and written along with a track hub. |
gtf |
a GTF file describing gene models in the case of analyzing a custom, non-directly supported organism. It will be converted to the .bigBed format and written along with a track hub. |
forceHub |
when |
rc |
Fraction of cores to use. |
A string with the link(s) to the created tracks.
Panagiotis Moulos
dataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) if (.Platform$OS.type == "unix") link <- createSignalTracks(file.path(path,"targets.txt"),"mm9")dataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) if (.Platform$OS.type == "unix") link <- createSignalTracks(file.path(path,"targets.txt"),"mm9")
This function creates false (or true) discovery
curves using a list containing several outputs
from diagplotFtd.
diagplotAvgFtd(ftdrObj, output = "x11", path = NULL, draw = TRUE, ...)diagplotAvgFtd(ftdrObj, output = "x11", path = NULL, draw = TRUE, ...)
ftdrObj |
a list with outputs from
|
output |
one or more R plotting
device to direct the plot result to.
Supported mechanisms: |
path |
the path to create output files. |
draw |
boolean to determine whether
to plot the curves or just return the
calculated values (in cases where the user
wants the output for later averaging
for example). Defaults to |
... |
further arguments to be passed to
plot devices, such as parameter from
|
A named list with two members: the first member
(avgFtdr) contains a list with the
means and the standard deviations of the averaged
ftdrObj and are used to create the plot.
The second member (path) contains the
path to the created figure graphic.
Panagiotis Moulos
p11 <- 0.001*matrix(runif(300),100,3) p12 <- matrix(runif(300),100,3) p21 <- 0.001*matrix(runif(300),100,3) p22 <- matrix(runif(300),100,3) p31 <- 0.001*matrix(runif(300),100,3) p32 <- matrix(runif(300),100,3) p1 <- rbind(p11,p21) p2 <- rbind(p12,p22) p3 <- rbind(p31,p32) rownames(p1) <- rownames(p2) <- rownames(p3) <- paste("gene",1:200,sep="_") colnames(p1) <- colnames(p2) <- colnames(p3) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20), rep(1,10),rep(2,10),rep(0,80)) names(truth) <- rownames(p1) ftdObj1 <- diagplotFtd(truth,p1,N=100,draw=FALSE) ftdObj2 <- diagplotFtd(truth,p2,N=100,draw=FALSE) ftdObj3 <- diagplotFtd(truth,p3,N=100,draw=FALSE) ftdObj <- list(ftdObj1,ftdObj2,ftdObj3) avgFtdObj <- diagplotAvgFtd(ftdObj)p11 <- 0.001*matrix(runif(300),100,3) p12 <- matrix(runif(300),100,3) p21 <- 0.001*matrix(runif(300),100,3) p22 <- matrix(runif(300),100,3) p31 <- 0.001*matrix(runif(300),100,3) p32 <- matrix(runif(300),100,3) p1 <- rbind(p11,p21) p2 <- rbind(p12,p22) p3 <- rbind(p31,p32) rownames(p1) <- rownames(p2) <- rownames(p3) <- paste("gene",1:200,sep="_") colnames(p1) <- colnames(p2) <- colnames(p3) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20), rep(1,10),rep(2,10),rep(0,80)) names(truth) <- rownames(p1) ftdObj1 <- diagplotFtd(truth,p1,N=100,draw=FALSE) ftdObj2 <- diagplotFtd(truth,p2,N=100,draw=FALSE) ftdObj3 <- diagplotFtd(truth,p3,N=100,draw=FALSE) ftdObj <- list(ftdObj1,ftdObj2,ftdObj3) avgFtdObj <- diagplotAvgFtd(ftdObj)
A wrapper over the general boxplot function, suitable for matrices produced and processed with the metaseqr package. Intended for internal use but can be easily used as stand-alone. It can colors boxes based on group depending on the name argument.
diagplotBoxplot(mat, name = NULL, logIt = "auto", yLim = "default", isNorm = FALSE, output = "x11", path = NULL, altNames = NULL, ...)diagplotBoxplot(mat, name = NULL, logIt = "auto", yLim = "default", isNorm = FALSE, output = "x11", path = NULL, altNames = NULL, ...)
mat |
the count data matrix. |
name |
the names of the samples plotted on the boxplot. See also Details. |
logIt |
whether to log transform the values of mat
or not. It can be |
yLim |
custom y-axis limits. Leave the string
|
isNorm |
a logical indicating whether object contains raw or normalized data. It is not essential and it serves only plot annotation purposes. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
altNames |
an optional vector of names, e.g. HUGO
gene symbols, alternative or complementary to the unique
rownames of |
... |
further arguments to be passed to plot
devices, such as parameter from |
Regarding name, if NULL, the function check
the column names of mat. If they are also
NULL, sample names are autogenerated. If
name="none", no sample names are plotted. If name
is a list, it should be the sampleList argument provided
to the manin metaseqr2 function. In that case, the boxes
are colored per group.
The filename of the boxplot produced if it's a file.
Panagiotis Moulos
# Non-normalized boxplot require(DESeq2) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) # Normalized boxplot normArgs <- getDefaults("normalization","deseq2") object <- normalizeDeseq2(dataMatrix,sampleList,normArgs) diagplotBoxplot(object,sampleList)# Non-normalized boxplot require(DESeq2) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) # Normalized boxplot normArgs <- getDefaults("normalization","deseq2") object <- normalizeDeseq2(dataMatrix,sampleList,normArgs) diagplotBoxplot(object,sampleList)
This function uses the read counts matrix to create heatmap or correlogram correlation plots.
diagplotCor(mat, type = c("heatmap", "correlogram"), output = "x11", path = NULL, ...)diagplotCor(mat, type = c("heatmap", "correlogram"), output = "x11", path = NULL, ...)
mat |
the read counts matrix or data frame. |
type |
create heatmap of correlogram plots. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filename of the pairwise comparisons plot produced if it's a file.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) diagplotCor(dataMatrix,type="heatmap") diagplotCor(dataMatrix,type="correlogram")dataMatrix <- metaseqR2:::exampleCountData(2000) diagplotCor(dataMatrix,type="heatmap") diagplotCor(dataMatrix,type="correlogram")
This function plots a heatmap of the differentially expressed genes produced by the metaseqr workflow, useful for quality control, e.g. whether samples belonging to the same group cluster together.
diagplotDeHeatmap(x, scale = c("asis", "zscore"), con = NULL, output = "x11", path = NULL, ...)diagplotDeHeatmap(x, scale = c("asis", "zscore"), con = NULL, output = "x11", path = NULL, ...)
x |
the data matrix to create a heatmap for. |
scale |
value scale in the heatmap. As provided
( |
con |
an optional string depicting a name (e.g. the contrast name) to appear in the title of the volcano plot. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filenames of the plots produced in a named list with
names the whichPlot argument. If
output="x11", no output filenames are produced.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" M <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(M,sampleList,contrast) diagplotDeHeatmap(dataMatrix[p[[1]]<0.05,])dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" M <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(M,sampleList,contrast) diagplotDeHeatmap(dataMatrix[p[[1]]<0.05,])
A wrapper around the plotting functions availale in the
EDASeq normalization Bioconductor package. For analytical
explanation of each plot please see the vignette of the
EDASeq package. It is best to use this function through
the main plotting function
metaseqrPlot.
diagplotEdaseq(x, sampleList, covar = NULL, isNorm = FALSE, whichPlot = c("meanvar", "meandiff", "gcbias", "lengthbias"), output = "x11", altNames = NULL, path = NULL, ...)diagplotEdaseq(x, sampleList, covar = NULL, isNorm = FALSE, whichPlot = c("meanvar", "meandiff", "gcbias", "lengthbias"), output = "x11", altNames = NULL, path = NULL, ...)
x |
the count data matrix. |
sampleList |
the list containing condition names and the samples under each condition. |
covar |
The covariate to plot counts against.
Usually |
isNorm |
a logical indicating whether object contains raw or normalized data. It is not essential and it serves only plot annotation purposes. |
whichPlot |
the EDASeq package plot to generate. See Details. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
altNames |
optional names, alternative or complementary
to the rownames of |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
Regarding whichPlot, it can be one or more of
"meanvar", "meandiff", "gcbias" or
"lengthbias". Please refer to the documentation of
the EDASeq package for details on the use of these
plots. The whichPlot="lengthbias" case is
not covered by EDASeq documentation, however it is
similar to the GC-bias plot when the covariate is the
gene length instead of the GC content.
The filenames of the plot produced in a named list with
names the which.plot argument. If output="x11", no
output filenames are produced.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotEdaseq(dataMatrix,sampleList,whichPlot="meandiff")dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotEdaseq(dataMatrix,sampleList,whichPlot="meandiff")
This function plots a grid of four graphs depicting: in the first row, the numbers of filtered genes per chromosome in the first column and per biotype in the second column. In the second row, the percentages of filtered genes per chromosome related to the whole genome in the first columns and per biotype in the second column.
diagplotFiltered(x, y, output = "x11", path = NULL, ...)diagplotFiltered(x, y, output = "x11", path = NULL, ...)
x |
an annotation data frame like the ones produced
by |
y |
an annotation data frame like the ones produced
by |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filenames of the plots produced in a named list with
names the which.plot argument. If
output="x11", no output filenames are produced.
Panagiotis Moulos
data("mm9GeneData",package="metaseqR2") y <- mm9GeneCounts[,c(1:6,8,7)] x <- y[-sample(1:nrow(y),1000),] diagplotFiltered(x,y)data("mm9GeneData",package="metaseqR2") y <- mm9GeneCounts[,c(1:6,8,7)] x <- y[-sample(1:nrow(y),1000),] diagplotFiltered(x,y)
This function creates false (or true) discovery
curves using a matrix of p-values (such a matrix
can be derived for example from the result table
of metaseqr2 by subsetting the table
to get the p-values from several algorithms)
given a ground truth vector for differential
expression.
diagplotFtd(truth, p, type = "fpc", N = 2000, output = "x11", path = NULL, draw = TRUE, ...)diagplotFtd(truth, p, type = "fpc", N = 2000, output = "x11", path = NULL, draw = TRUE, ...)
truth |
the ground truth differential
expression vector. It should contain only
zero and non-zero elements, with zero denoting
non-differentially expressed genes and non-zero,
differentially expressed genes. Such a vector
can be obtained for example by using the
|
p |
a p-value matrix whose rows correspond
to each element in the |
type |
what to plot, can be |
N |
create the curves based on the
top (or bottom) |
output |
one or more R plotting device to
direct the plot result to. Supported mechanisms:
|
path |
the path to create output files. |
draw |
boolean to determine whether to
plot the curves or just return the calculated
values (in cases where the user wants the
output for later averaging for example).
Defaults to |
... |
further arguments to be passed to plot
devices, such as parameter from |
A named list with two members: the first member
(ftdr) contains the values used to create
the plot. The second member (path) contains
the path to the created figure graphic.
Panagiotis Moulos
p1 <- 0.001*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20), rep(1,10),rep(2,10),rep(0,80)) names(truth) <- rownames(p) ftdObj <- diagplotFtd(truth,p,N=100)p1 <- 0.001*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20), rep(1,10),rep(2,10),rep(0,80)) names(truth) <- rownames(p) ftdObj <- diagplotFtd(truth,p,N=100)
Creates a Multi-Dimensional Scale plot for the given samples based on the count data matrix. MDS plots are very useful for quality control as you can easily see of samples of the same groups are clustered together based on the whole dataset.
diagplotMds(x, sampleList, method = "spearman", logIt = TRUE, output = "x11", path = NULL, ...)diagplotMds(x, sampleList, method = "spearman", logIt = TRUE, output = "x11", path = NULL, ...)
x |
the count data matrix. |
sampleList |
the list containing condition names and the samples under each condition. |
method |
which correlation method to use. Same as
the method parameter in |
logIt |
whether to log transform the values of x or not. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filename of the MDS plot produced if it's a file.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotMds(dataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotMds(dataMatrix,sampleList)
A wrapper around the plotting functions availale in the
NOISeq Bioconductor package. For analytical explanation
of each plot please see the vignette of the NOISeq
package. It is best to use this function through the
main plotting function metaseqrPlot.
diagplotNoiseq(x, sampleList, covars, whichPlot = c("biodetection", "countsbio", "saturation", "rnacomp", "readnoise", "biodist"), output = "x11", biodistOpts = list(p = NULL, pcut = NULL, name = NULL), path = NULL, isNorm = FALSE, ...)diagplotNoiseq(x, sampleList, covars, whichPlot = c("biodetection", "countsbio", "saturation", "rnacomp", "readnoise", "biodist"), output = "x11", biodistOpts = list(p = NULL, pcut = NULL, name = NULL), path = NULL, isNorm = FALSE, ...)
x |
the count data matrix. |
sampleList |
the list containing condition names and the samples under each condition. |
covars |
a list (whose annotation elements are
ideally a subset of an annotation data frame produced by
|
whichPlot |
the NOISeq package plot to generate. See Details |
biodistOpts |
a list with the following members: p
(a vector of p-values, e.g. the p-values of a contrast),
pcut (a unique number depicting a p-value cutoff,
required for the |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
isNorm |
a logical indicating whether object contains raw or normalized data. It is not essential and it serves only plot annotation purposes. |
... |
further arguments to be passed to plot
devices, such as parameter from |
Regarding whichPlot, It can be one or more of
"biodetection", "countsbio",
"saturation", "rnacomp", "readnoise"
or "biodist". Please refer to the documentation of
the NOISeq package for details on the use of these plots.
The whichPlot="saturation" case is modified to be
more informative by producing two kinds of plots.
The filenames of the plots produced in a named list with
names the whichPlot argument. If
output="x11", no output filenames are produced.
Please note that in case of "biodist" plots, the
behavior of the function is unstable, mostly due to the
very specific inputs this plotting function accepts in
the NOISeq package. We have tried to predict unstable
behavior and avoid exceptions through the use of tryCatch
but it's still possible that you might run onto an error.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths covars <- list( data=dataMatrix, length=lengths, gc=runif(nrow(dataMatrix)), chromosome=data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts, end=ends ), factors=data.frame(class=metaseqR2:::asClassVector(sampleList)), biotype=c(rep("protein_coding",nrow(dataMatrix)/2),rep("ncRNA", nrow(dataMatrix)/2)) ) p <- runif(nrow(dataMatrix)) diagplotNoiseq(dataMatrix,sampleList,covars=covars, biodistOpts=list(p=p,pcut=0.1,name="A_vs_B"))dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths covars <- list( data=dataMatrix, length=lengths, gc=runif(nrow(dataMatrix)), chromosome=data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts, end=ends ), factors=data.frame(class=metaseqR2:::asClassVector(sampleList)), biotype=c(rep("protein_coding",nrow(dataMatrix)/2),rep("ncRNA", nrow(dataMatrix)/2)) ) p <- runif(nrow(dataMatrix)) diagplotNoiseq(dataMatrix,sampleList,covars=covars, biodistOpts=list(p=p,pcut=0.1,name="A_vs_B"))
This function uses the read counts matrix to create pairwise correlation plots. The upper diagonal of the final image contains simple scatterplots of each sample against each other (log2 scale) while the lower diagonal contains mean-difference plots for the same samples (log2 scale). This type of diagnostic plot may not be interpretable for more than 10 samples.
diagplotPairs(x, output = "x11", altNames = NULL, path = NULL, ...)diagplotPairs(x, output = "x11", altNames = NULL, path = NULL, ...)
x |
the read counts matrix or data frame. |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
altNames |
optional names, alternative or complementary
to the rownames of |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filename of the pairwise comparisons plot produced if it's a file.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) diagplotPairs(dataMatrix)dataMatrix <- metaseqR2:::exampleCountData(2000) diagplotPairs(dataMatrix)
This function creates basic ROC curves using a
matrix of p-values (such a matrix can be
derived for example from the result table of
metaseqr2 by subsetting the table
to get the p-values from several algorithms)
given a ground truth vector for differential
expression and a significance level.
diagplotRoc(truth, p, sig = 0.05, x = "fpr", y = "tpr", output = "x11", path = NULL, draw = TRUE, ...)diagplotRoc(truth, p, sig = 0.05, x = "fpr", y = "tpr", output = "x11", path = NULL, draw = TRUE, ...)
truth |
the ground truth differential
expression vector. It should contain only
zero and non-zero elements, with zero denoting
non-differentially expressed genes and non-zero,
differentially expressed genes. Such a vector
can be obtained for example by using the
|
p |
a p-value matrix whose rows correspond
to each element in the |
sig |
a significance level (0 < |
x |
what to plot on x-axis, can be one of
|
y |
what to plot on y-axis, same as
|
output |
one or more R plotting device to
direct the plot result to. Supported mechanisms:
|
path |
the path to create output files. |
draw |
boolean to determine whether to
plot the curves or just return the calculated
values (in cases where the user wants the
output for later averaging for example).
Defaults to |
... |
further arguments to be passed to plot
devices, such as parameter from |
A named list with two members. The first member is
a list containing the ROC statistics: TP
(True Postives), FP (False Positives),
FN (False Negatives), TN
(True Negatives), FPR (False Positive Rate),
FNR (False Negative Rate), TPR (True
Positive Rate), TNR (True Negative Rate),
AUC (Area Under the Curve). The second is
the path to the created figure graphic.
Panagiotis Moulos
p1 <- 0.001*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20),rep(1,10), rep(2,10),rep(0,80)) names(truth) <- rownames(p) rocObj <- diagplotRoc(truth,p)p1 <- 0.001*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") truth <- c(rep(1,40),rep(-1,40),rep(0,20),rep(1,10), rep(2,10),rep(0,80)) names(truth) <- rownames(p) rocObj <- diagplotRoc(truth,p)
This function uses the R package VennDiagram and plots an up to 5-way Venn diagram depicting the common and specific to each statistical algorithm genes, for each contrast. Mostly for internal use because of its main argument which is difficult to construct, but can be used independently if the user grasps the logic.
diagplotVenn(pmat, fcmat = NULL, pcut = 0.05, fcut = 0.5, direction = c("dereg", "up", "down"), nam = as.character(round(1000 * runif(1))), output = "x11", path = NULL, altNames = NULL, ...)diagplotVenn(pmat, fcmat = NULL, pcut = 0.05, fcut = 0.5, direction = c("dereg", "up", "down"), nam = as.character(round(1000 * runif(1))), output = "x11", path = NULL, altNames = NULL, ...)
pmat |
a matrix with p-values corresponding to the application of each statistical algorithm. See also Details. |
fcmat |
an optional matrix with fold changes corresponding to the application of each statistical algorithm. See also Details. |
pcut |
if |
fcut |
a p-value cutoff for statistical
significance. Defaults to |
direction |
if |
nam |
a name to be appended to the output graphics
file (if |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. If
|
altNames |
an optional named vector of names, e.g.
HUGO gene symbols, alternative or complementary to the
unique gene names which are the rownames of |
... |
further arguments to be passed to plot
devices, such as parameter from |
Regarding pmat, the p-value matrix must have the
colnames attribute and the colnames should correspond to
the name of the algorithm used to fill the specific
column (e.g. if "statistics"=c("deseq","edger",
"nbpseq") then colnames(pmat) <-
c("deseq","edger","nbpseq").
Regarding fcmat, the fold change matrix must have
the colnames attribute and the colnames should correspond
to the name of the algorithm used to fill the specific
column (see the parameter pmat).
Regarding direction, it can be one of "dereg"
for the total of regulated genes, where
abs(fcmat[,n])>=fcut (default), "up" for
the up-regulated genes where fcmat[,n]>=fcut or
"down" for the up-regulated genes where
fcmat[,n]<=-fcut.
The filenames of the plots produced in a named list with
names the which.plot argument. If
output="x11", no output filenames are produced.
Panagiotis Moulos
require(VennDiagram) p1 <- 0.01*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") vennContents <- diagplotVenn(p)require(VennDiagram) p1 <- 0.01*matrix(runif(300),100,3) p2 <- matrix(runif(300),100,3) p <- rbind(p1,p2) rownames(p) <- paste("gene",1:200,sep="_") colnames(p) <- paste("method",1:3,sep="_") vennContents <- diagplotVenn(p)
This function plots a volcano plot or returns a JSON string which is used to render aninteractive in case of HTML reporting.
diagplotVolcano(f, p, con = NULL, fcut = 1, pcut = 0.05, altNames = NULL, output = "x11", path = NULL, ...)diagplotVolcano(f, p, con = NULL, fcut = 1, pcut = 0.05, altNames = NULL, output = "x11", path = NULL, ...)
f |
the fold changes which are to be plotted on the x-axis. |
p |
the p-values whose -log10 transformation is going to be plotted on the y-axis. |
con |
an optional string depicting a name (e.g. the contrast name) to appear in the title of the volcano diagplot. |
fcut |
a fold change cutoff so as to draw two vertical lines indicating the cutoff threshold for biological significance. |
pcut |
a p-value cutoff so as to draw a horizontal line indicating the cutoff threshold for statistical significance. |
altNames |
an optional vector of names, e.g. HUGO
gene symbols, alternative or complementary to the unique
names of |
output |
one or more R plotting device to direct the
plot result to. Supported mechanisms: |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
The filenames of the plots produced in a named list with
names the which.plot argument. If
output="x11", no output filenames are produced.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" M <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(M,sampleList,contrast) ma <- apply(M[,sampleList$A],1,mean) mb <- apply(M[,sampleList$B],1,mean) f <- log2(ifelse(mb==0,1,mb)/ifelse(ma==0,1,ma)) diagplotVolcano(f,p[[1]],con=contrast) #j <- diagplotVolcano(f,p[[1]],con=contrast,output="json")dataMatrix <- metaseqR2:::exampleCountData(5000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" M <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(M,sampleList,contrast) ma <- apply(M[,sampleList$A],1,mean) mb <- apply(M[,sampleList$B],1,mean) f <- log2(ifelse(mb==0,1,mb)/ifelse(ma==0,1,ma)) diagplotVolcano(f,p[[1]],con=contrast) #j <- diagplotVolcano(f,p[[1]],con=contrast,output="json")
This function downsamples the library sizes of a read counts table to the lowest library size, according to the methdology used in (Soneson and Delorenzi, BMC Bioinformatics, 2013).
downsampleCounts(counts)downsampleCounts(counts)
counts |
the read counts table which is subjected to downsampling. |
The downsampling process involves random sampling.
For guaranteed reproducibility, be sure to use
set.seed before downsampling. By default,
when the metaseqR2 package is loaded, the seed is
set to 42.
The downsampled counts matrix.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(5000) D <- downsampleCounts(dataMatrix)dataMatrix <- metaseqR2:::exampleCountData(5000) D <- downsampleCounts(dataMatrix)
This function automatically estimates weights
for the "weight" and "dperm_weight"
options of metaseqR2 for combining p-values from
multiple statistical tests. It creates simulated
dataset based on real data and then performs
statistical analysis with metaseqR2 several times
in order to derive False Discovery Curves. Then,
the average areas under the false discovery curves
are used to construct weights for each algorithm,
according to its performance when using simulated
data.
estimateAufcWeights(counts, normalization, statistics, nsim = 10, N = 10000, samples = c(3, 3), ndeg = c(500, 500), top = 500, modelOrg = "mm9", fcBasis = 1.5, drawFpc = FALSE, rc = NULL, ...)estimateAufcWeights(counts, normalization, statistics, nsim = 10, N = 10000, samples = c(3, 3), ndeg = c(500, 500), top = 500, modelOrg = "mm9", fcBasis = 1.5, drawFpc = FALSE, rc = NULL, ...)
counts |
the real raw counts table from which the simulation parameters will be estimated. It must not be normalized and must contain only integer counts, without any other annotation elements and unique gene identifiers as the rownames attribute. |
normalization |
same as |
statistics |
same as |
nsim |
the number of simulations to perform to estimate the weights. It default to 10. |
N |
the number of genes to produce.
See |
samples |
a vector with 2 integers, which are the number of samples for each condition (two conditions currently supported). |
ndeg |
a vector with 2 integers, which are the number of differentially expressed genes to be produced. The first element is the number of up-regulated genes while the second is the number of down-regulated genes. |
fcBasis |
the minimum fold-change for deregulation. |
top |
the top |
modelOrg |
the organism from which the
data are derived. It must be one of
|
drawFpc |
draw the averaged false
discovery curves? Default to |
rc |
the fraction of the available cores to use in a multicore system. |
... |
Further arguments to be passed to
|
The weight estimation process involves a lot of
random sampling. For guaranteed reproducibility,
be sure to use set.seed prior to any
calculations. By default, when the metaseqR2 package
is loaded, the seed is set to 42.
A vector of weights to be used in
metaseqr2 with the
weights option.
Panagiotis Moulos
require(zoo) data("mm9GeneData",package="metaseqR2") weights <- estimateAufcWeights( counts=as.matrix(mm9GeneCounts[sample(nrow(mm9GeneCounts),1000),9:12]), normalization="edaseq", statistics=c("edger","limma"), nsim=1,N=100,ndeg=c(10,10),top=10,modelOrg=NULL, rc=0.01,libsizeGt=1e+5 )require(zoo) data("mm9GeneData",package="metaseqR2") weights <- estimateAufcWeights( counts=as.matrix(mm9GeneCounts[sample(nrow(mm9GeneCounts),1000),9:12]), normalization="edaseq", statistics=c("edger","limma"), nsim=1,N=100,ndeg=c(10,10),top=10,modelOrg=NULL, rc=0.01,libsizeGt=1e+5 )
This function reads a read counts table containing real RNA-Seq data (preferebly with more than 20 samples so as to get as much accurate as possible estimations) and calculates a population of count means and dispersion parameters which can be used to simulate an RNA-Seq dataset with synthetic genes by drawing from a negative binomial distribution. This function works in the same way as described in (Soneson and Delorenzi, BMC Bioinformatics, 2013) and (Robles et al., BMC Genomics, 2012).
estimateSimParams(realCounts, libsizeGt = 3e+6, rowmeansGt = 5,eps = 1e-11, rc = NULL, draw = FALSE)estimateSimParams(realCounts, libsizeGt = 3e+6, rowmeansGt = 5,eps = 1e-11, rc = NULL, draw = FALSE)
realCounts |
a text tab-delimited file with real RNA-Seq data. See Details. |
libsizeGt |
a library size below which
samples are excluded from parameter estimation
(default: |
rowmeansGt |
a row means (mean counts over samples for each gene) below which genes are excluded from parameter estimation (default: 5). |
eps |
the tolerance for the convergence
of |
rc |
in case of parallel optimization, the fraction of the available cores to use. |
draw |
boolean to determine whether to
plot the estimated simulation parameters
(mean and dispersion) or not. Defaults to
|
Regarding realCounts, the file should strictly
contain a unique gene name (e.g. Ensembl accession) in
the first column and all other columns should contain
read counts for each gene. Each column must be named
with a unique sample identifier. See examples in the
ReCount database
http://bowtie-bio.sourceforge.net/recount/.
Also, the parameter estimation involves a lot of
random sampling. For guaranteed reproducibility,
be sure to use set.seed prior to any
calculations. By default, when the metaseqR2 package
is loaded, the seed is set to 42.
A named list with two members: muHat
which contains negative binomial mean
estimates and phiHat which contains
dispersion estimates.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) parList <- estimateSimParams(dataMatrix,libsizeGt=3e+4)dataMatrix <- metaseqR2:::exampleCountData(2000) parList <- estimateSimParams(dataMatrix,libsizeGt=3e+4)
For Ensembl based annotations, this function connects to the
EBI's Biomart service using the package biomaRt and downloads
annotation elements (gene co-ordinates, exon co-ordinates,
gene identifications, biotypes etc.) for each of the supported
organisms. For UCSC/RefSeq annotations, it connects to the
respective SQL databases if the package RMySQL is
present, otherwise it downloads flat files and build a
temporary SQLite database to make the necessaru build
queries. See the help page of metaseqr2
for a list of supported organisms.
getAnnotation(org, type, refdb = "ensembl", ver = NULL, rc = NULL)getAnnotation(org, type, refdb = "ensembl", ver = NULL, rc = NULL)
org |
the organism for which to download annotation (one of the supported ones). |
type |
|
refdb |
the online source to use to fetch
annotation. It can be |
ver |
the version of the annotation to use. |
rc |
Fraction of cores to use. Same as the
|
A data frame with the canonical (not isoforms!) genes or
exons of the requested organism. When
type="genes", the data frame has the following
columns: chromosome, start, end, gene_id, gc_content,
strand, gene_name, biotype. When type="exon" the
data frame has the following columns: chromosome, start,
end, exon_id, gene_id, strand, gene_name, biotype. When
type="utr" the data frame has the following columns:
chromosome, start, end, transcript_id, gene_id, strand,
gene_name, biotype. The gene_id and exon_id correspond to
Ensembl, UCSC or RefSeq gene, transcript and exon accessions
respectively. The gene_name corresponds to HUGO nomenclature
gene names.
The data frame that is returned contains only "canonical" chromosomes for each organism. It does not contain haplotypes or random locations and does not contain chromosome M.
Panagiotis Moulos
mm10Genes <- getAnnotation("mm10","gene")mm10Genes <- getAnnotation("mm10","gene")
This function returns a list with the default settings for each filtering, statistical and normalization algorithm included in the metaseqR package. See the documentation of the main function and the documentation of each statistical and normalization method for details.
getDefaults(what, method = NULL)getDefaults(what, method = NULL)
what |
a keyword determining the procedure for which
to fetch the default settings according to method
parameter. It can be one of |
method |
the supported algorithm included in metaseqR for which to fetch the default settings. Se Details. |
When what is "normalization", method is
one of "edaseq", "deseq", "edger",
"noiseq" or "nbpseq". When what is
"statistics", method is one of "deseq",
"edger", "noiseq", "limma",
or "nbpseq". When method is
"biotypeFilter", what is the input
organism (see the main metaseqr2 help page
for a list of supported organisms).
A list with default setting that can be used directly in the call of metaseqr.
Panagiotis Moulos
normArgsEdaseq <- getDefaults("normalization","edaseq") statArgsEdger <- getDefaults("statistics","edger")normArgsEdaseq <- getDefaults("normalization","edaseq") statArgsEdger <- getDefaults("statistics","edger")
This function returns a data frame with information on locally installed, supported or custom, annotations.
getInstalledAnnotations(obj = NULL)getInstalledAnnotations(obj = NULL)
obj |
|
The function returns a data.frame object with
the installed local annotations.
Panagiotis Moulos
db <- file.path(system.file(package="metaseqR2"), "annotation.sqlite") if (file.exists(db)) ig <- getInstalledAnnotations(obj=db)db <- file.path(system.file(package="metaseqR2"), "annotation.sqlite") if (file.exists(db)) ig <- getInstalledAnnotations(obj=db)
This function returns pre-calculated weights
for human, chimpanzee, mouse, fruitfly and
arabidopsis based on the performance of
simulated datasets estimated from real data
from the ReCount database
(http://bowtie-bio.sourceforge.net/recount/).
Currently pre-calculated weights are available only
when all six statistical tests are used and for
normalization with EDASeq. For other combinations,
use the estimateAufcWeights function.
getWeights(org = c("human", "chimpanzee", "mouse", "fruitfly", "arabidopsis", "rat"))getWeights(org = c("human", "chimpanzee", "mouse", "fruitfly", "arabidopsis", "rat"))
org |
|
A named vector of convex weights.
Panagiotis Moulos
wh <- getWeights("human")wh <- getWeights("human")
This data set contains p-values calculated with each of the supported statistical testing algorithms in metaseqR2 for 1000 genes. The purpose of this matrix is to demonstrate the p-value combination methods as well as be used for a playground for other such methods and with other metaseqR2 facilities.
a matrix with p-values from metaseqR2 supported tests.
Panagiotis Moulos
Giakountis et al. (https://doi.org/10.1016/j.celrep.2016.05.038)
This function creates a local annotation database to be used with metaseqr2 so as to avoid long time on the fly annotation downloads and formatting.
importCustomAnnotation(gtfFile, metadata, level = c("gene", "transcript", "exon"), type = c("gene", "exon", "utr"))importCustomAnnotation(gtfFile, metadata, level = c("gene", "transcript", "exon"), type = c("gene", "exon", "utr"))
gtfFile |
a GTF file containing the gene structure of the organism to be imported. |
metadata |
a list with additional information about
the annotation to be imported. The same as in the
|
level |
same as the |
type |
same as the |
The function returns a GenomicRanges object with
the requested annotation.
Panagiotis Moulos
# Dummy GTF as example chromInfo <- data.frame(length=c(1000L,2000L,1500L), row.names=c("A","B","C")) # Build with the metadata list filled (you can also provide a version) myGenes <- importCustomAnnotation( gtfFile=file.path(system.file(package="metaseqR2"),"dummy.gtf"), metadata=list( organism="dummy", source="dummy_db", version=1, chromInfo=chromInfo ), level="gene",type="gene" ) ## Real data! ## Gene annotation dump from Ensembl #download.file(paste0("ftp://ftp.ensembl.org/pub/release-98/gtf/", # "dasypus_novemcinctus/Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # file.path(tempdir(),"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz")) ## Build with the metadata list filled (you can also provide a version) #dasGenes <- importCustomAnnotation( # gtfFile=file.path(tempdir(),"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # metadata=list( # organism="dasNov3_test", # source="ensembl_test" # ), # level="gene",type="gene" #)# Dummy GTF as example chromInfo <- data.frame(length=c(1000L,2000L,1500L), row.names=c("A","B","C")) # Build with the metadata list filled (you can also provide a version) myGenes <- importCustomAnnotation( gtfFile=file.path(system.file(package="metaseqR2"),"dummy.gtf"), metadata=list( organism="dummy", source="dummy_db", version=1, chromInfo=chromInfo ), level="gene",type="gene" ) ## Real data! ## Gene annotation dump from Ensembl #download.file(paste0("ftp://ftp.ensembl.org/pub/release-98/gtf/", # "dasypus_novemcinctus/Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # file.path(tempdir(),"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz")) ## Build with the metadata list filled (you can also provide a version) #dasGenes <- importCustomAnnotation( # gtfFile=file.path(tempdir(),"Dasypus_novemcinctus.Dasnov3.0.98.gtf.gz"), # metadata=list( # organism="dasNov3_test", # source="ensembl_test" # ), # level="gene",type="gene" #)
The library size list for mm9GeneCounts. See the
data set description.
a named list with library sizes.
Panagiotis Moulos
ENCODE (http://genome.ucsc.edu/encode/)
This function creates loads an annotation element from
the local annotation database to be used with metaseqr2.
If the annotation is not found and the organism is
supported, the annotation is created on the fly but not
imported in the local database. Use
buildAnnotationDatabase for this purpose.
loadAnnotation(genome, refdb, level = c("gene", "transcript", "exon"), type = c("gene", "exon", "utr"), version="auto", db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), summarized = FALSE, asdf = FALSE, rc = NULL)loadAnnotation(genome, refdb, level = c("gene", "transcript", "exon"), type = c("gene", "exon", "utr"), version="auto", db = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), summarized = FALSE, asdf = FALSE, rc = NULL)
genome |
a |
refdb |
a |
level |
same as the |
type |
same as the |
version |
same as the |
db |
same as the |
summarized |
if |
asdf |
return the result as a |
rc |
same as the |
The function returns a GenomicRanges object with
the requested annotation.
Panagiotis Moulos
db <- file.path(system.file(package="metaseqR2"), "annotation.sqlite") if (file.exists(db)) gr <- loadAnnotation(genome="hg19",refdb="ensembl", level="gene",type="gene",db=db)db <- file.path(system.file(package="metaseqR2"), "annotation.sqlite") if (file.exists(db)) gr <- loadAnnotation(genome="hg19",refdb="ensembl", level="gene",type="gene",db=db)
This function creates simulated RNA-Seq gene expression datasets using the method presented in (Soneson and Delorenzi, BMC Bioinformatics, 2013). For the time being, it creates only simulated datasets with two conditions.
makeSimDataSd(N, param, samples = c(5, 5), ndeg = rep(round(0.1*N), 2), fcBasis = 1.5, libsizeRange = c(0.7, 1.4), libsizeMag = 1e+7, modelOrg = NULL, simLengthBias = FALSE)makeSimDataSd(N, param, samples = c(5, 5), ndeg = rep(round(0.1*N), 2), fcBasis = 1.5, libsizeRange = c(0.7, 1.4), libsizeMag = 1e+7, modelOrg = NULL, simLengthBias = FALSE)
N |
the number of genes to produce. |
param |
a named list with negative binomial
parameter sets to sample from. The first member is
the mean parameter to sample from ( |
samples |
a vector with 2 integers, which are the number of samples for each condition (two conditions currently supported). |
ndeg |
a vector with 2 integers, which are the number of differentially expressed genes to be produced. The first element is the number of up-regulated genes while the second is the number of down-regulated genes. |
fcBasis |
the minimum fold-change for deregulation. |
libsizeRange |
a vector with 2 numbers
(generally small, see the default), as they
are multiplied with |
libsizeMag |
a (big) number to multiply
the |
modelOrg |
the organism from which the
real data are derived from. It must be one
of the supported organisms (see the main
|
simLengthBias |
a boolean to instruct the simulator to create genes whose read counts is proportional to their length. This is achieved by sorting in increasing order the mean parameter of the negative binomial distribution (and the dispersion according to the mean) which will cause an increasing gene count length with the sampling. The sampled lengths are also sorted so that in the final gene list, shorter genes have less counts as compared to the longer ones. The default is FALSE. |
The simulated data generation involves a lot of
random sampling. For guaranteed reproducibility,
be sure to use set.seed prior to any
calculations. By default, when the metaseqR2 package
is loaded, the seed is set to 42.
A named list with two members. The first
member (simdata) contains the
synthetic dataset
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) ## File "bottomly_read_counts.txt" from the ReCount database #download.file(paste("http://bowtie-bio.sourceforge.net/recount/", # "countTables/bottomly_count_table.txt",sep=""), # destfile="~/bottomly_count_table.txt") N <- 2000 #parList <- estimateSimParams("~/bottomly_read_counts.txt") parList <- estimateSimParams(dataMatrix,libsizeGt=3e+4) sim <- makeSimDataSd(N,parList) synthData <- sim$simdata trueDeg <- which(sim$truedeg!=0)dataMatrix <- metaseqR2:::exampleCountData(2000) ## File "bottomly_read_counts.txt" from the ReCount database #download.file(paste("http://bowtie-bio.sourceforge.net/recount/", # "countTables/bottomly_count_table.txt",sep=""), # destfile="~/bottomly_count_table.txt") N <- 2000 #parList <- estimateSimParams("~/bottomly_read_counts.txt") parList <- estimateSimParams(dataMatrix,libsizeGt=3e+4) sim <- makeSimDataSd(N,parList) synthData <- sim$simdata trueDeg <- which(sim$truedeg!=0)
This function creates simulated RNA-Seq gene expression
datasets using the simulateReadCounts function
from the Bioconductor package TCC and it adds simulated
annoation elements. For further information please
consult the TCC package documentation.
makeSimDataTcc(...)makeSimDataTcc(...)
... |
parameters to the |
A list with the following members: simdata holding
the simulated dataset complying with metaseqr2
requirements, and simparam holding the simulation
parameters (see TCC documentation). Note that the produced
data are based in an Arabidopsis dataset.
Panagiotis Moulos
if (require(TCC)) { dd <- makeSimDataTcc(Ngene=1000,PDEG=0.2, DEG.assign=c(0.9,0.1), DEG.foldchange=c(5,5),replicates=c(3,3)) head(dd$simdata) }if (require(TCC)) { dd <- makeSimDataTcc(Ngene=1000,PDEG=0.2, DEG.assign=c(0.9,0.1), DEG.foldchange=c(5,5),replicates=c(3,3)) head(dd$simdata) }
This function is the main metaseqr2 workhorse and implements the main metaseqr2 workflow which performs data read, filtering, normalization and statistical selection, creates diagnostic plots and exports the results and a report if requested. The metaseqr2 function is responsible for assembling all the steps of the metaseqr2 pipeline which i) reads the input gene or exon read count table ii) performs prelimininary filtering of data by removing chrM and other non-essential information for a typical differential gene expression analysis as well as a preliminary expression filtering based on the exon counts, if an exon read count file is provided. iii) performs data normalization with one of currently widely used algorithms, including EDASeq (Risso et al., 2011), DESeq (Anders and Huber, 2010), edgeR (Robinson et al., 2010), NOISeq (Tarazona et al., 2012) or no normalization iv) performs a second stage of filtering based on the normalized gene expression according to several gene filters v) performs statistical testing with one or more of currently widely used algorithms, including DESeq (Anders and Huber, 2010), edgeR (Robinson et al., 2010), NOISeq (Tarazona et al., 2012), limma (Smyth et al., 2005) for RNA-Seq data vi) in the case of multiple statistical testing algorithms, performs meta-analysis using one of six vailable methods (see the meta.p argument) vii) exports the resulting differentially expressed gene list in text tab-delimited format viii) creates a set of diagnostic plots either available in the aforementioned packages or metaseqr2 specific ones and ix) creates a comprehensive HTML report which summarizes the run information, the results and the diagnostic plots. Certain diagnostic plots (e.g. the volcano plot) can be interactive with the use of the external Highcharts (http://www.highcharts.com) JavaScript library for interactive graphs. Although the inputs to the metaseqr2 workflow are many, in practice, setting only very few of them and accepting the defaults as the rest can result in quite comprehensible results for mainstream organisms like mouse, human, fly and rat.
metaseqr2(counts, sampleList, excludeList = NULL, fileType = c("auto", "sam", "bam", "bed"), path = NULL, contrast = NULL, libsizeList = NULL, embedCols = list(idCol = 4, gcCol = NA, nameCol = NA, btCol = NA), annotation = NULL, org = c("hg18", "hg19", "hg38", "mm9", "mm10", "rn5", "rn6", "dm3", "dm6", "danrer7", "pantro4", "susscr3", "tair10", "equcab2"), refdb = c("ensembl", "ucsc", "refseq"), version = "auto", transLevel = c("gene", "transcript", "exon"), countType = c("gene", "exon","utr"), utrOpts = list(frac = 1, minLength = 300, downstream = 50), exonFilters = list(minActiveExons = list(exonsPerGene = 5, minExons = 2, frac = 1/5)), geneFilters = list(length = list(length = 500), avgReads = list(averagePerBp = 100, quantile = 0.25), expression = list(median = TRUE, mean = FALSE, quantile = NA, known = NA, custom = NA), biotype = getDefaults("biotypeFilter", org[1]), presence = list(frac = 0.25, minCount = 10, perCondition = FALSE)), whenApplyFilter = c("postnorm", "prenorm"), normalization = c("deseq", "deseq2", "edaseq", "edger", "noiseq", "nbpseq", "absseq", "dss", "each", "none"), normArgs = NULL, statistics = c("deseq", "deseq2", "edger", "noiseq", "limma", "nbpseq", "absseq", "dss"), statArgs = NULL, adjustMethod = sort(c(p.adjust.methods, "qvalue")), metaP = if (length(statistics) > 1) c("simes", "bonferroni", "fisher", "dperm_min", "dperm_max", "dperm_weight", "fperm", "whitlock", "minp", "maxp", "weight", "pandora", "none") else "none", weight = rep(1/length(statistics), length(statistics)), nperm = 10000, pcut = NA, logOffset = 1, pOffset = NULL, preset = NULL, qcPlots = c("mds", "biodetection", "countsbio", "saturation", "readnoise","filtered", "correl", "pairwise", "boxplot", "gcbias", "lengthbias", "meandiff", "meanvar", "rnacomp", "deheatmap", "volcano", "biodist", "mastat", "statvenn", "foldvenn", "deregulogram"), figFormat = c("png", "jpg", "tiff", "bmp", "pdf", "ps"), outList = FALSE, exportWhere = NA, exportWhat = c("annotation", "p_value", "adj_p_value", "meta_p_value", "adj_meta_p_value", "fold_change", "stats", "counts","flags"), exportScale = c("natural", "log2", "log10", "vst", "rpgm"), exportValues = c("raw", "normalized"), exportStats = c("mean", "median", "sd", "mad", "cv", "rcv"), exportCountsTable = FALSE, restrictCores = 0.6, report = TRUE, reportTop = 0.1, reportTemplate = "default", saveGeneModel = TRUE, verbose = TRUE, runLog = TRUE, reportDb = c("dexie", "sqlite"), localDb = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), offlineReport = TRUE, createTracks = FALSE, overwriteTracks = FALSE, trackExportPath = file.path(exportWhere, "tracks"), trackInfo = list(stranded = FALSE, normTo = 1e+9, urlBase = "http://www.trackserver.me", fasta = NULL, gtf = NULL, hubInfo = list(name = "MyHub", shortLabel = "My hub", longLabel = "My hub long", email = "[email protected]")), .progressFun = NULL, .exportR2C = FALSE,...)metaseqr2(counts, sampleList, excludeList = NULL, fileType = c("auto", "sam", "bam", "bed"), path = NULL, contrast = NULL, libsizeList = NULL, embedCols = list(idCol = 4, gcCol = NA, nameCol = NA, btCol = NA), annotation = NULL, org = c("hg18", "hg19", "hg38", "mm9", "mm10", "rn5", "rn6", "dm3", "dm6", "danrer7", "pantro4", "susscr3", "tair10", "equcab2"), refdb = c("ensembl", "ucsc", "refseq"), version = "auto", transLevel = c("gene", "transcript", "exon"), countType = c("gene", "exon","utr"), utrOpts = list(frac = 1, minLength = 300, downstream = 50), exonFilters = list(minActiveExons = list(exonsPerGene = 5, minExons = 2, frac = 1/5)), geneFilters = list(length = list(length = 500), avgReads = list(averagePerBp = 100, quantile = 0.25), expression = list(median = TRUE, mean = FALSE, quantile = NA, known = NA, custom = NA), biotype = getDefaults("biotypeFilter", org[1]), presence = list(frac = 0.25, minCount = 10, perCondition = FALSE)), whenApplyFilter = c("postnorm", "prenorm"), normalization = c("deseq", "deseq2", "edaseq", "edger", "noiseq", "nbpseq", "absseq", "dss", "each", "none"), normArgs = NULL, statistics = c("deseq", "deseq2", "edger", "noiseq", "limma", "nbpseq", "absseq", "dss"), statArgs = NULL, adjustMethod = sort(c(p.adjust.methods, "qvalue")), metaP = if (length(statistics) > 1) c("simes", "bonferroni", "fisher", "dperm_min", "dperm_max", "dperm_weight", "fperm", "whitlock", "minp", "maxp", "weight", "pandora", "none") else "none", weight = rep(1/length(statistics), length(statistics)), nperm = 10000, pcut = NA, logOffset = 1, pOffset = NULL, preset = NULL, qcPlots = c("mds", "biodetection", "countsbio", "saturation", "readnoise","filtered", "correl", "pairwise", "boxplot", "gcbias", "lengthbias", "meandiff", "meanvar", "rnacomp", "deheatmap", "volcano", "biodist", "mastat", "statvenn", "foldvenn", "deregulogram"), figFormat = c("png", "jpg", "tiff", "bmp", "pdf", "ps"), outList = FALSE, exportWhere = NA, exportWhat = c("annotation", "p_value", "adj_p_value", "meta_p_value", "adj_meta_p_value", "fold_change", "stats", "counts","flags"), exportScale = c("natural", "log2", "log10", "vst", "rpgm"), exportValues = c("raw", "normalized"), exportStats = c("mean", "median", "sd", "mad", "cv", "rcv"), exportCountsTable = FALSE, restrictCores = 0.6, report = TRUE, reportTop = 0.1, reportTemplate = "default", saveGeneModel = TRUE, verbose = TRUE, runLog = TRUE, reportDb = c("dexie", "sqlite"), localDb = file.path(system.file(package = "metaseqR2"), "annotation.sqlite"), offlineReport = TRUE, createTracks = FALSE, overwriteTracks = FALSE, trackExportPath = file.path(exportWhere, "tracks"), trackInfo = list(stranded = FALSE, normTo = 1e+9, urlBase = "http://www.trackserver.me", fasta = NULL, gtf = NULL, hubInfo = list(name = "MyHub", shortLabel = "My hub", longLabel = "My hub long", email = "[email protected]")), .progressFun = NULL, .exportR2C = FALSE,...)
counts |
a text tab-delimited file containing gene,
exon or 3'UTR counts in one of the following formats: i) the
first column contains unique gene or exon identifiers and
the rest of the columns contain the read counts for each
sample. ii) The first n columns should contain only **gene**
annotation elements like chromosomal locations, gene
accessions, exon accessions, GC content etc. and the rest
columns should contain gene read counts, iii) |
sampleList |
a list containing condition names and the samples under each condition or a small tab-delimited file with the experiment description. Not needed when restoring a previous analysis. See Details for analytical description. |
excludeList |
a list of samples to exclude, in the
same (list) format as |
path |
an optional path where all the BED/BAM files are placed, to be prepended to the BAM/BED file names in the targets file. See Details for further information. |
fileType |
the type of raw input files. It can be
|
contrast |
a character vector of contrasts to be
tested in the statistical testing step(s) of the pipeline.
Each element of contrast should STRICTLY have the format
"ConditionA_vs_ConditionB_vs_...". Special attention is
needed as fold change calculations are based on this
argument. If it is |
libsizeList |
an optional named list where names
represent samples (MUST be the same as the samples in
|
embedCols |
a named list with column numbers to guide the case of embedded annotation. See Details for further information. |
annotation |
It can be one of i) |
org |
the supported organisms by |
refdb |
the reference annotation repository from
which to retrieve annotation elements to use with
metaseqr2. It can be one of |
version |
the version of the annotation to use. See Details. |
transLevel |
perform differential expression
analysis at which transcriptional unit, can be one of
|
countType |
the type of reads inside the counts
file. It can be one of |
utrOpts |
a named list with members |
exonFilters |
a named list whose names are the names of the supported exon filters and its members the filter parameters. See section "Exon filters" below for details. |
geneFilters |
a named list whose names are the names of the supported gene filters and its members the filter parameters. See section "Gene filters" below for details. |
whenApplyFilter |
a character string determining
when to apply the exon and/or gene filters, relative to
normalization. It can be |
normalization |
the normalization algorithm to be
applied on the count data. It can be one of
|
normArgs |
a named list whose names are the names
of the normalization algorithm parameters and its members
parameter values. See section "Normalization parameters"
below for details. Leave |
statistics |
one or more statistical analyses to be
performed by the metaseqr2 pipeline. It can be one or more
of |
statArgs |
a named list whose names are the names
of the statistical algorithms used in the pipeline. Each
member is another named list whose names are the
algorithm parameters and its members are the parameter
values. See section "Statistics parameters" below for
details. Leave |
adjustMethod |
the multiple testing p-value
adjustment method. It can be one of
|
metaP |
the meta-analysis method to combine
p-values from multiple statistical tests . It can be
one of |
weight |
a vector of weights with the same length as
the |
nperm |
the number of permutations performed to
derive the meta p-value when |
pcut |
a p-value cutoff for exporting differentially genes, default is to export all the non-filtered genes. |
logOffset |
an offset to be added to values during
logarithmic transformations in order to avoid Infinity
(default is |
pOffset |
a value between |
preset |
an analysis strictness preset.
|
qcPlots |
a set of diagnostic plots to show/create.
It can be one or more of |
figFormat |
the format of the output diagnostic
plots. It can be one or more of |
outList |
a logical controlling whether to export a list with the results in the running environment. |
exportWhere |
an output directory for the project results (report, lists, diagnostic plots etc.) |
exportWhat |
the content of the final lists. It can
be one or more of |
exportScale |
export values from one or more
transformations applied to the data. It can be one or
more of |
exportValues |
It can be one or more of
|
exportStats |
calculate and export several
statistics on raw and normalized counts, condition-wise.
It can be one or more of |
exportCountsTable |
exports also the calculated
read counts table when input is read from bam files
and exports also the normalized count table in all
cases. Defaults to |
restrictCores |
in case of parallel execution of
several subfunctions, the fraction of the available cores
to use. In some cases if all available cores are used
( |
report |
a logical value controlling whether to
produce a summary report or not. Defaults to
|
reportTop |
a fraction of top statistically
significant genes to append to the HTML report. This
helps in keeping the size of the report as small as
possible, as appending the total gene list might
create a huge HTML file. Users can always retrieve
the whole gene lists from the report links. Defaults
to |
reportTemplate |
an HTML template to use for the report. Do not change this unless you know what you are doing. |
saveGeneModel |
in case of exon analysis, a list
with exon counts for each gene will be saved to the file
|
verbose |
print informative messages during
execution? Defaults to |
runLog |
write a log file of the |
reportDb |
database system to use for storing the
report intereactive graphs. Can be |
localDb |
the metaseqR2 annotaation database location.
See also |
offlineReport |
|
.
createTracks |
option to create normalized bigWig
files to display in a genome browser (e.g. UCSC). Defaults
to |
overwriteTracks |
overwrite tracks if they already
exist? Defaults to |
trackExportPath |
where to export the bigWig files,
defaults to |
trackInfo |
if |
.progressFun |
a function which updates a
|
.exportR2C |
export additional RData along with
|
... |
further arguments that may be passed to
plotting functions, related to |
When counts is a tab-delimited file, the following
restrictions apply:
In the case (i) the first cell of each row is a
gene or exon accession and the rest are integers
representing the counts for that accession. In that
case, the annotation parameter should strictly
be NULL or an external file in GTF format.
In the case (ii) the annotation parameter
can also be "embedded". The ideal embedded
annotation contains 8 columns, chromosome, gene or
exon start, gene or exon end, gene or exon accession,
GC-content (fraction or percentage), strand, HUGO
gene symbol and gene biotype (e.g. "protein_coding"
or "ncRNA"). When the annotation parameter is
"embedded", certain of these features are
mandatory (co-ordinates and accessions). If they are
not present, the pipeline will not run. If additional
elements are not present (e.g. GC content or biotypes),
certain features of metaseqr2 will not be available.
For example, EDASeq normalization will not be
performed based on a GC content covariate but based
on gene length which is not what the authors of
EDASeq suggest. If biotypes are not present, a lot of
diagnostic plots will not be available. If the HUGO
gene symbols are missing, the final annotation will
contain only gene accessions and thus be less
comprehensible. Counts can be a data frame satisfying
the above conditions. It is a data frame by default
when read2count is used.
In the case (iii) the .RData file
(output of save function contains
static input elements (list containing the gene
model (exon counts for each gene), gene and exon
annotation to avoid re-(down)loading and/or gene
counts depending on countType). This kind of
input facilitates the re-analysis of the same
experiment, using different filtering, normalization
and statistical algorithms. This .RData file
is produced when saveGeneModel=TRUE.
In the case (iv) counts can be a list
representing the gene model (exon/UTR counts for each
gene). This .RData file can be generated by
setting saveGeneModel=TRUE when performing data
analysis for the first time.
Regarding sampleList it should have the format
sampleList <- list(ConditionA=c("Sample_A1",
"Sample_A2","Sample_A3"),
ConditionB=c("Sample_B1","Sample_B2"),
ConditionC=c("Sample_C1","Sample_C2")). The names of
the samples in list members MUST match the column names
containing the read counts in the counts file. If they do
not match, the pipeline will either crash or at best, ignore
several of your samples. Alternative, sampleList can
be a small tab-delimited file structured as follows: the
first line of the external tab delimited file should contain
column names (names are not important). The first column MUST
contain UNIQUE sample names and the second column MUST
contain the biological condition where each of the samples
in the first column should belong to. If the counts
argument is missing, the sampleList argument MUST be
a targets text tab-delimited file which contains the sample
names, the BAM/BED file names and the biological
conditions/groups for each sample/file. The file should be
text tab-delimited and structured as follows: the first line
of the external tab delimited file should contain column
names (names are not important). The first column MUST
contain UNIQUE sample names. The second column MUST contain
the raw BAM/BED files WITH their full path. Alternatively,
the path argument should be provided. If path
is not provided and if the files in the second column of
the targets file do not contain a path to a directory, the
current directory is assumed to be the BAM/BED file
container. The third column MUST contain the biological
condition where each of the samples in the first column
should belong to.
Regarding contrast, a valid example based on the
sampleList above is
contrast <- c("ConditionA_vs_ConditionB",
"ConditionA_vs_ConditionC",
"ConditionA_vs_ConditionB_vs_ConditionC"). The
first element of pairwise contrasts (e.g. "ConditionA"
above) MUST be the control condition or any reference
that ConditionB is checked against. metaseqr2 uses this
convention to properly calculate fold changes.
Regarding embedCols, this list must contain four
members, named idCol, gcCol, nameCol
and btCol, which hold the position in the delimited
file with embedded annotation, where unique gene ids, GC
content, gene names and gene biotypes respectively are
located. More specifically:
idCol is an integer denoting the column
number in the file (or data frame) provided with the
counts argument, where the unique gene accessions are.
Default to 4 which is the standard feature name
column in a BED file.
gcCol is an integer denoting the column
number in the file (or data frame) provided with the
counts argument, where each gene's GC content
is given. If not provided, GC content normalization
provided by EDASeq will not be available.
nameCol is an integer denoting the column
number in the file (or data frame) provided with the
counts argument, where the HUGO gene symbols are given.
If not provided, it will not be available when reporting
results. In addition, the "known" gene filter
will not be available for application.
btCol is an integer denoting the column
number in the file (or data frame) provided with the
counts argument, where the gene biotypes are given.
If not provided, the "biodetection",
"countsbio", "saturation",
"filtered" and "biodist" plots will not
be available.
Regarding annotation instructs metaseqr2 where to find the
annotation for the given counts file. It can be one of i)
"download" (default) for automatic downloading of
the annotation for the organism specified by the org
parameter (using biomaRt), ii) "embedded" if the
annotation elements are embedded in the read counts file
or iv) a file specified by the user which should be as
similar as possible to the "download" case, in
terms of column structure.
Regarding org, it can be, for human genomes
"hg18", "hg19" or "hg38", for mouse
genomes "mm9", "mm10", for rat genomes
"rn5" or "rn6", for drosophila genome
"dm3" or "dm6", for zebrafish genome
"danrer7", "danrer10" or "danrer11",
for chimpanzee genome "pantro4", "pantro5",
for pig genome "susscr3", "susscr11", for
Arabidopsis thaliana genome "tair10" and for
Equus caballus genome "equcab2". Finally, it can
be "USER_NAMED_ORG" with a custom organism which
has been imported to the annotation database by the user
using a GTF file. For example org="mm10_p1".
Regarding version, this is an integer denoting the
version of the annotation to use from the local annotation
database or fetch on the fly. For Ensembl, it corresponds
to Ensembl releases, while for UCSC/RefSeq, it is the
date of creation (locally).
Regarding whenApplyFilter, in the case of
whenApplyFilter="prenorm", a first normalization
round is applied to a copy of the gene counts matrix in
order to derive the proper normalized values that will
constitute the several expression-based filtering
cutoffs.
Regarding metaP, for the "fisher" and
"fperm" methods, see the documentation of the R
package MADAM. For the "whitlock" method, see the
documentation of the survcomp Bioconductor package. With
the "maxp" option, the final p-value is the maximum
p-value out of those returned by each statistical test.
This is equivalent to an "intersection" of the results
derived from each algorithm so as to have a final list with
the common genes returned by all statistical tests.
Similarly, when meta.p="minp", is equivalent to a
"union" of the results derived from each algorithm so as
to have a final list with all the genes returned by all
statistical tests. The latter can be used as a very lose
statistical threshold to aggregate results from all methods
regardless of their False Positive Rate. With the
"simes" option, the method proposed by Simes
(Simes, R. J., 1986) is used. With the "dperm_min",
"dperm.max", "dperm.weight" options, a
permutation procedure is initialed, where nperm
permutations are performed across the samples of the
normalized counts matrix, producing nperm permuted
instances of the initital dataset. Then, all the chosen
statistical tests are re-executed for each permutation.
The final p-value is the number of times that the p-value
of the permuted datasets is smaller than the original
dataset. The p-value of the original dataset is created
based on the choice of one of dperm.min,
dperm.max or dperm.weight options. In case of
dperm.min, the intial p-value vector is consists
of the minimum p-value resulted from the applied
statistical tests for each gene. The maximum p-value
is used with the dperm.max option. With the
dperm.weight option, the weight
weighting vector for each statistical test is used to
weight each p-value according to the power of
statistical tests (some might work better for a
specific dataset). Be careful as the permutation
procedure usually requires a lot of time. However,
it should be the most accurate. This method will NOT
work when there are no replicated samples across
biological conditions. In that case, use
meta.p="simes" instead. Finally, there are the
"minp", "maxp" and "weight"
options which correspond to the latter three methods
but without permutations. Generally, permutations
would be accurate to use when the experiment includes
>5 samples per condition (or even better 7-10) which
is rather rare in RNA-Seq experiments. Finally,
"pandora" is the same as "weight" and is
added to be in accordance with the main algorithm name.
Regarding pOffset, it is used to correct for
the case of a p-value which is equal to 0 as a result
of internal numerical and approximation procedures.
When NULL, random numbers greater than 0 and
less than or equal to 0.5 are used to multiply the
offending p-values with the lowest provided non-zero
p-value, maintaining thus a virtual order of
significance, avoiding having the same p-values for
two tests and assuming that all zero p-values represent
extreme statistical significance. When a numeric
between 0 and 1, this number is used for the above
multiplication instead.
Regarding qcPlots The "mds" stands
for Mutlti-Dimensional Scaling and it creates a PCA-like
plot but using the MDS dimensionality reduction instead.
It has been succesfully used for NGS data (e.g. see the
package htSeqTools) and it shows how well samples from
the same condition cluster together. For
"biodetection", "countsbio",
"saturation", "rnacomp",
"readnoise", "biodist" see the vignette of
NOISeq package. The "saturation" case has been
rewritten in order to display more samples in a more
simple way. In addition, the "readnoise" plots
represent an older version or the RNA composition plot
included in older versions of NOISeq. For "gcbias",
"lengthbias", "meandiff", "meanvar"
see the vignette of EDASeq package. "lenghtbias"
is similar to "gcbias" but using the gene length
instead of the GC content as covariate. The "boxplot"
option draws boxplots of log2 transformed gene counts. The
"filtered" option draws a 4-panel figure with the
filtered genes per chromosome and per biotype, as
absolute numbers and as fractions of the genome. See also
the help page of diagplotFiltered. The
"deheatmap" option performs hierarchical
clustering and draws a heatmap of differentially
expressed genes. In the context of diagnostic plots, it's
useful to see if samples from the same groups cluster
together after statistical testing. The "volcano"
option draws a volcano plot for each contrast and if a
report is requested, an interactive volcano plot is
presented in the HTML report. The "venn" option
will draw an up to 5-way Venn diagram depicting the
common and specific to each statistical algorithm genes
and for each contrast, when meta-analysis is performed.
The "correl" option creates two correlation
graphs: the first one is a correlation heatmap (a
correlation matrix which depicts all the pairwise
correlations between each pair of samples in the counts
matrix is drawn as a clustered heatmap) and the second
one is a correlogram plot, which summarizes the
correlation matrix in the form of ellipses (for an
explanation please see the vignette/documentation of the
R package corrplot. Set qcPlots=NULL if you don't
want any diagnostic plots created.
Regarding reportDb, contrary with the first version of
metaseqR, all graphs in the metaseqR2 report are interactive
with the usage of the JavaScript libraries Highcharts, plotly
(heatmaply) and jvenn.js. However, this adds a great burden
regarding rendering the final HTML file and its content,
a burden which becomes heavier by the fact the metaseqR2
report is rendered using knitr and rmarkdown
instead of raw HTML (previously, brew). Therefore, the
pre-calculated JSON objects representing the graphs are
stored either in a report-specific IndexedDB
(https://javascript.info/indexeddb) flavor called Dexie
(https://dexie.org/) (default) or in an SQLite database
and then queried using sql.js
(https://github.com/kripken/sql.js/). Dexie is
prefered because it is very efficient and can produce an
independent report that does not need to be served through
a web-server and can be viewed locally. Although Dexie is
very efficient, some caution is required as knitr and
render from rmarkdown are not very
memory efficient when rendering larger HTML files. A large
HTML file may be produced when analyzing a large dataset
with a lot of contrasts that may result in a lot of tables.
In such cases, if the report generation crashes with errors
related to memory, try lowering the reportTop
argument. reportTop does not affect the final lists
of differentially expressed genes, only the report tables.
The same must be applied also if the report takes too much
time to load. If the report is to be served through a web
server like Apache (e.g. when the report is provided by a
facility to end users), reportDb="sqlite" may be
preferred as the total report size will be smaller because
of an SQLite database hosting all plots which are queried
when required but from the SQLite database and not from
the in-browser database (Dexie). **Note** that when using
an SQLite database, you will **NOT** be able to view the
report in any browser other than Microsoft Edge because
of security policies regarding local file access. **Note**
also that sql.js is a rather large JavaScript library
(around 2.5MB).
Regarding trackInfo, it is a helper list to guide the
bigWig track creation and has the following members:
stranded, which can be TRUE or
FALSE depending on whether you wish to create
stranded tracks by separating + and - strand reads.
In the case of stranded tracks, a UCSC Genome Brower
trackhub is created. Individual tracks can be retrieved
from the trackhub.
normTo, which is a large integer, denoting
the total sum of signal to be used as the normalization
target. It defaults to 1e+9. This means that if
for a particular sample the sum of signal is 1.5e+9
(sum(sapply(coverage(x),sum)) == 1.5e+9) then
this is linearly scaled to 1e+9.
urlBase, which is a base url appended to
the bigWig files produced (the base path of the
bigDataUrl in UCSC Genome Browser track lines).
hubInfo, a list with the track hub
description in case of stranded tracks. Please see the
track hub specifications at the UCSC Genome Browser site.
fasta, reference genome in FASTA format for
the case of analyzing a custom, non-directly supported
organism. It will be converted to the .2bit format and
written along with a track hub.
gtf, a GTF file describing gene models in
the case of analyzing a custom, non-directly supported
organism. It will be converted to the .bigBed format and
written along with a track hub. Essentially the same as
annotation$gtf.
All files (bigWig files, track/trackhub info) are written in
the tracks subdirectory of the main path where the
report and the outputs are written.
If outList is TRUE, a named list whose
length is the same as the number of requested contrasts.
Each list member is named according to the corresponding
contrast and contains a data frame of differentially
expressed genes for that contrast. The contents of the
data frame are defined by the exportWhat,
exportScale, exportStats, exportValues parameters. If
report is TRUE, the output list contains
two main elements. The first is described above (the
analysis results) and the second contains the same
results but in HTML formatted tables.
The exon filters are a set of filters which are applied
after the gene models are assembled from the read counts
of individual exons and before the gene expression is
summarized from the exons belonging to each gene. These
filters can be applied when the input read counts file
contains exon reads. It is not applicable when the input
file already contains gene counts. Such filters can be
for example "accept genes where all the exons contain
more than x reads" or "accept genes where there is read
presence in at least m/n exons, n being the total exons
of the gene". Such filters are NOT meant for detecting
differential splicing as also the whole metaseqr2
pipeline, thus they should not be used in that context.
The exonFilters argument is a named list of
filters, where the names are the filter names and the
members are the filter parameters (named lists with
parameter name, parameter value). See the usage of the
metaseqr2 function for an example of how these
lists are structured. The supported exon filters in the
current version are: i) minActiveExons which
implements a filter for demanding m out of n exons of a
gene to have a certain read presence with parameters
exonsPerGene, minExons and frac.
The filter is described as follows: if a gene has up to
exonsPerGene exons, then read presence is
required in at least minExons of them, else read
presence is required in a frac fraction of the
total exons. With the default values, the filter
instructs that if a gene has up to 5 exons, read presence
is required in at least 2, else in at least 20
exons, in order to be accepted. More filters will be
implemented in future versions and users are encouraged
to propose exon filter ideas to the author by mail. See
metaseqr2 usage for the defaults. Set
exonFilters=NULL to not apply any exon filtering.
The gene filters are a set of filters applied to gene
expression as this is manifested through the read
presence on each gene and are preferably applied after
normalization. These filters can be applied both when the
input file or data frame contains exon read counts and
gene read counts. Such filter can be for example "accept
all genes above a certain count threshold" or "accept all
genes with expression above the median of the normalized
counts distribution" or "accept all with length above a
certain threshold in kb" or "exclude the 'pseudogene'
biotype from further analysis". The supported gene
filters in the current version, which have the same
structure as the exon filters (named list of lists with
filter names, parameter names and parameter arguments)
are: i) length which implements a length filter
where genes are accepted for further analysis if they are
above length (its parameter) kb. ii)
avg.reads which implements a filter where a gene
is accepted for further analysis if it has more average
reads than the quantile of the average count
distribution per averagePerBp base pairs. In
summary, the reads of each gene are averaged per
averagePerBp based on each gene's length (in
case of exons, input the "gene's length" is the sum of
the lengths of exons) and the quantile quantile of
the average counts distribution is calculated for each
sample. Genes passing the filter should have an average
read count larger than the maximum of the vector of the
quantiles calculated above. iii) expression which
implements a filter based on the overall expression of a
gene. The parameters of this filter are: median,
where genes below the median of the overall count
distribution are not accepted for further analysis (this
filter has been used to distinguish between "expressed"
and "not expressed" genes in several cases, e.g. (Mokry
et al., NAR, 2011) with a logical as value, mean
which is the same as median but using the mean,
quantile which is the same as the previous two but
using a specific quantile of the total counts
distribution, known, where in this case, a set of
known not-expressed genes in the system under
investigation are used to estimate an expression cutoff.
This can be quite useful, as the genes are filtered based
on a "true biological" cutoff instead of a statistical
cutoff. The value of this filter is a character vector of
HUGO gene symbols (MUST be contained in the annotation,
thus it's better to use annotation="download")
whose counts are used to build a "null" expression
distribution. The 90th quantile of this distribution is
then the expression cutoff. This filter can be combined
with any other filter. Be careful with gene names as they
are case sensitive and must match exactly ("Pten" is
different from "PTEN"!). iv) biotype where in this
case, genes with a certain biotype (MUST be contained in
the annotation, thus it's better to use
annotation="download") are excluded from the
analysis. This filter is a named list of logical, where
names are the biotypes in each genome and values are
TRUE or FALSE. If the biotype should be
excluded, the value should be TRUE else
FALSE. See the result of
get.defaults("biotype.filter","hg19") for an
example. Finally, in future versions there will be
support for user-defined filters in the form of a
function. v) presence where in this case, a gene
is further considered for statistical testing if
frac (x100 for a percentage value) have more
than minCount reads across all samples
(perCondition=FALSE) or across the samples
of each condition (perCondition=TRUE).
The normalization parameters are passed again as a named
list where the names of the members are the normalization
parameter names and the values are the normalization
parameter values. You should check the documentation of
the packages EDASeq, DESeq, edgeR, NOISeq and NBPSeq for
the parameter names and parameter values. There are a few
exceptions in parameter names: in case of
normalization="edaseq" the only parameter names
are within.which and between.which,
controlling the withing lane/sample and between
lanes/samples normalization algorithm. In the case
of normalization="nbpseq", there is one
additional parameter called main.method which can
take the calues "nbpseq" or "nbsmyth".
These values correspond to the two different workflows
available in the NBPSeq package. Please, consult the
NBPSeq package documentation for further details. For the
rest of the algorithms, the parameter names are the same
as the names used in the respective packages. For
examples, please use the getDefaults
function.
The statistics parameters as passed to statistical
algorithms in metaseqr2, exactly with the same way as the
normalization parametes above. In this case, there is one
more layer in list nesting. Thus, statArgs is a
named list whose names are the names the algorithms used
(see the statistics parameter). Each member is
another named list,with parameters to be used for each
statistical algorithm. Again, the names of the member
lists are parameter names and the values of the member
lists are parameter values. You should check the
documentations of DESeq, edgeR, NOISeq, limma and
NBPSeq for these parameters. There are a few exceptions
in parameter names: In case of statistics="edger",
apart from the rest of the edgeR statistical testing
arguments, there is the argument mainMethod which
can be either "classic" or "glm", again
defining whether the binomial test or GLMs will be used
for statistical testing. For examples, please use the
getDefaults function. When
statistics="nbpseq", apart from the rest arguments
of the NBPSeq functions estimate.disp and
estimate.dispersion, there is the argument
mainMethod which can be "nbpseq" or
"nbsmyth". This argument determines the parameters
to be used by the estimate.dispersion function or
by the estimate.disp function to estimate RNA-Seq
count dispersions. The difference between the two is that
they constitute different starting points for the two
workflows in the package NBPSeq. The first worklfow (with
mainMethod="nbpseq" and the
estimate.dispersion function is NBPSeq package
specific, while the second (with
mainMethod="nbsmyth" and the estimate.disp
function is similar to the workflow of the edgeR package.
For additional information regarding the statistical
testing in NBPSeq, please consult the documentation of
the NBPSeq package.
The analysis presets are a set of keywords (only one can
be used) that predefine some of the parameters of the
metaseqr2 pipeline. For the time being they are quite
simple and they control i) the strictness of filtering
and statistical thresholding with three basic levels
("all", "medium", "strict") and ii) the data columns that
are exported, again in three basic ways ("basic",
"normal", "full") controlling the amount of data to be
exported. These keywords can be combined with a dot in
the middle (e.g. "all.basic" to define an analysis
preset. When using analysis presets, the following
argumentsof metaseqr2 are overriden: exonFilters,
geneFilters, pcut, exportWhat,
exportScale, exportValues,
exonStats. If you want to explicitly control the
above arguments, the preset argument should be set
to NULL (default). Following is a synopsis of the
different presets and the values of the arguments they
moderate:
"all_basic": use all genes (do not filter)
and export all genes and basic annotation and statistics
elements. In this case, the above described arguments become:
exonFilters=NULL
geneFilters=NULL
pcut=1
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean")
"all_normal": use all genes (do not filter)
and export all genes and normal annotation and statistics
elements. In this case, the above described arguments
become:
exonFilters=NULL
geneFilters=NULL
pcut=1
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change","stats",
"counts")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean","sd","cv")
"all_full": use all genes (do not filter)
and export all genes and all annotation and statistics
elements. In this case, the above described arguments
become:
exonFilters=NULL
geneFilters=NULL
pcut=1
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change","stats","counts")
exportScale=c("natural","log2","log10","vst")
exportValues=c("raw","normalized")
exportStats=c("mean","median","sd","mad",
"cv","rcv")
"medium_basic": apply a medium set of filters
and and export statistically significant genes and basic
annotation and statistics elements. In this case, the above
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=5,minExons=2,frac=1/5))
geneFilters=list(length=list(length=500),
avgReads=list(averagePerBp=100,quantile=0.25),
expression=list(median=TRUE,mean=FALSE,quantile=NA,
known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.05
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean")
"medium_normal": apply a medium set of filters
and export statistically significant genes and normal
annotation and statistics elements. In this case, the
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=5,minExons=2,frac=1/5))
geneFilters=list(length=list(length=500),
avgReads=list(averagePerBp=100,quantile=0.25),
expression=list(median=TRUE,mean=FALSE,
quantile=NA,known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.05
export.what=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change",
"stats","counts")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean","sd","cv")
"medium_full": apply a medium set of filters
and export statistically significant genes and full
annotation and statistics elements. In this case, the
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=5,minExons=2,frac=1/5))
geneFilters=list(length=list(length=500),
avgReads=list(averagePerBp=100,quantile=0.25),
expression=list(median=TRUE,mean=FALSE,
quantile=NA,known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.05
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change",
"stats","counts")
exportScale=c("natural","log2","log10",
"vst")
exportValues=c("raw","normalized")
exportStats=c("mean","median","sd","mad",
"cv","rcv")
"strict_basic": apply a strict set of filters
and and export statistically significant genes and basic
annotation and statistics elements. In this case, the
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=4,minExons=2,frac=1/4))
geneFilters=list(length=list(length=750),
avgReads=list(averagePerBp=100,quantile=0.5),
expression=list(median=TRUE,mean=FALSE,
quantile=NA,known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.01
exportWhat=c("annotation","p_value",
"adj_p_value","meta.p.value",
"adj_meta_p_value","fold_change")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean")
"strict_normal": apply a strict set of filters
and export statistically significant genes and normal
annotation and statistics elements. In this case, the
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=4,minExons=2,frac=1/4))
geneFilters=list(length=list(length=750),
avgReads=list(averagePerBp=100,quantile=0.5),
expression=list(median=TRUE,mean=FALSE,
quantile=NA,known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.01
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change",
"stats","counts")
exportScale=c("natural","log2")
exportValues=c("normalized")
exportStats=c("mean","sd","cv")
"strict_full": apply a strict set of filters
and export statistically significant genes and full
annotation and statistics elements. In this case, the
above described arguments become:
exonFilters=list(minActiveExons=
list(exonsPerGene=4,minExons=2,frac=1/4))
geneFilters=list(length=list(length=750),
avgReads=list(averagePerBp=100,quantile=0.5),
expression=list(median=TRUE,mean=FALSE,
quantile=NA,known=NA,custom=NA),
biotype=getDefaults("biotypeFilter",org[1]))
pcut=0.01
exportWhat=c("annotation","p_value",
"adj_p_value","meta_p_value",
"adj_meta_p_value","fold_change",
"stats","counts")
exportScale=c("natural","log2","log10",
"vst")
exportValues=c("raw","normalized")
exportStats=c("mean","median","sd","mad",
"cv","rcv")
Currently only gene and exon annotation from Ensembl
(http://www.ensembl.org), UCSC and RefSeq are supported.
In addition, the user may choose to use own GTF file on
the fly or import to the backend annotation database (see
buildAnnotationDatabase). Thus, the unique
gene ids in the counts files should correspond to valid
Ensembl, UCSC or RefSeq gene or exon accessions for the
organism of interest, or according to the user's GTF.
If you are not sure about the source of your counts file or
do not know how to produce it, it's better to start from the
original BAM/BED files (metaseqr2 will use the
read2count function to create a counts
file). Keep in mind that in the case of BED files, the
performance will be significantly lower and the overall
running time significanlty higher as the R functions
which are used to read BED files to proper structures
(GenomicRanges) and calculate the counts are quite slow.
An alternative way is maybe the easyRNASeq package
(Delhomme et al, 2012). The read2count
function does not use this package but rather makes use
of standard Bioconductor functions to handle NGS data. If
you wish to work outside R, you can work with other
popular read counters such as the HTSeq read counter
(http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html).
Please also note that in the current version, the members
of the geneFilters and exonFilters lists
are not checked for validity so be careful to supply with
correct names otherwise the pipeline will crash or at the
best case scenario, will ignore the filters. Also note
that when you are supplying metaseqr2 wtih an exon counts
table, gene annotation is always downloaded so please be
sure to have a working internet connection. In addition
to the above, if you have a multiple core system, be very
careful on how you are using the restrictCores
argument and generally how many cores you are using with
scripts purely written in R. The analysis with exon read
data can very easily cause memory problems, so unless you
have more than 64Gb of RAM available, consider setting
restrict.cores to something like 0.2 when working with
exon data. Finally, if you do not wish to download the
same annotation again and again when performing multiple
analyses, it is best to use the
buildAnnotationDatabase function to download
and store the resulting data frames in local SQLite
database and then use these files with the
org, refdb and version options.
Please note that the meta-analysis feature provided by metaseqr2 does not satisfy the strict definition of "meta-analysis", which is the combination of multiple similar datasets under the same statistical methodology. Instead it is the use of mulitple statistical tests applied to the same data. For the Simes method, please consult also "Simes, R. J. (1986). "An improved Bonferroni procedure for multiple tests of significance". Biometrika 73 (3): 751–754."
Also, if weight="meta_perm" ideally one would
want to create the same set of indices for a given
dataset so as to create reproducible p-values. To
achieve this, use the set.seed function prior to
any calculations. When metaseqR2 is loaded, the random
seed is set to 42.
Panagiotis Moulos
# An example pipeline with gene counts data("mm9GeneData",package="metaseqR2") result <- metaseqr2( counts=mm9GeneCounts, sampleList=sampleListMm9, contrast=c("adult_8_weeks_vs_e14.5"), libsizeList=libsizeListMm9, annotation="embedded", org="mm9", countType="gene", normalization="edger", statistics="edger", pcut=0.05, figFormat="png", qcPlots="mds", exportWhat=c("annotation","p_value","adj_p_value","fold_change"), exportScale="natural", exportValues="normalized", exportStats="mean", exportWhere=file.path(tempdir(),"test1"), restrictCores=0.01, geneFilters=list( length=list( length=500 ), avgReads=list( averagePerBp=100, quantile=0.25 ), expression=list( median=TRUE, mean=FALSE, quantile=NA, known=NA, custom=NA ), biotype=getDefaults("biotypeFilter","mm9") ), outList=TRUE ) head(result$data[["adult_8_weeks_vs_e14.5"]])# An example pipeline with gene counts data("mm9GeneData",package="metaseqR2") result <- metaseqr2( counts=mm9GeneCounts, sampleList=sampleListMm9, contrast=c("adult_8_weeks_vs_e14.5"), libsizeList=libsizeListMm9, annotation="embedded", org="mm9", countType="gene", normalization="edger", statistics="edger", pcut=0.05, figFormat="png", qcPlots="mds", exportWhat=c("annotation","p_value","adj_p_value","fold_change"), exportScale="natural", exportValues="normalized", exportStats="mean", exportWhere=file.path(tempdir(),"test1"), restrictCores=0.01, geneFilters=list( length=list( length=500 ), avgReads=list( averagePerBp=100, quantile=0.25 ), expression=list( median=TRUE, mean=FALSE, quantile=NA, known=NA, custom=NA ), biotype=getDefaults("biotypeFilter","mm9") ), outList=TRUE ) head(result$data[["adult_8_weeks_vs_e14.5"]])
This is the main function for producing sructured quality control and informative graphs base on the results of the various steps of the metaseqR package. The graphs produced span a variety of issues like good sample reproducibility (Multi-Dimensional Scaling plot, biotype detection, heatmaps. diagplotMetaseqr, apart from implementing certain package-specific plots, is a wrapper around several diagnostic plots present in other RNA-Seq analysis packages such as EDASeq and NOISeq.
metaseqrPlot(object, sampleList, annotation = NULL, contrastList = NULL, pList = NULL, thresholds = list(p = 0.05, f = 1), plotType = c("mds", "biodetection", "countsbio", "saturation", "readnoise", "rnacomp", "correl", "pairs", "boxplot", "gcbias", "lengthbias", "meandiff", "meanvar", "deheatmap", "volcano", "biodist", "filtered", "mastat", "deregulogram", "statvenn", "foldvenn"), isNorm = FALSE, output = "x11", path = NULL, ...)metaseqrPlot(object, sampleList, annotation = NULL, contrastList = NULL, pList = NULL, thresholds = list(p = 0.05, f = 1), plotType = c("mds", "biodetection", "countsbio", "saturation", "readnoise", "rnacomp", "correl", "pairs", "boxplot", "gcbias", "lengthbias", "meandiff", "meanvar", "deheatmap", "volcano", "biodist", "filtered", "mastat", "deregulogram", "statvenn", "foldvenn"), isNorm = FALSE, output = "x11", path = NULL, ...)
object |
a matrix or a data frame containing count data derived before or after the normalization procedure, filtered or not by the metaseqR2's filters and/or p-value. |
sampleList |
the list containing condition names and the samples under each condition. |
annotation |
a data frame containing annotation elements for each row in object. See also Details. |
contrastList |
the vector of contrasts as defined in
the main help page of |
pList |
a list of p-values for each contrast as
obtained from any of the |
thresholds |
a list with the elements |
plotType |
one or more of the diagnostic plots supported in metaseqR2 package. See also Details. |
isNorm |
a logical indicating whether object contains raw or normalized data. It is not essential and it serves only plot annotation purposes. |
output |
one or more R plotting device to direct the plot result to. See Details. |
path |
the path to create output files. |
... |
further arguments to be passed to plot
devices, such as parameter from |
Regarding object, the object can be fed to any of
the diagplotMetaseqr plotting systems but not every
plot is meaningful. For example, it's meaningless to
create a "biodist" plot for a count matrix before
normalization or statistical testing.
Regarding annotation, usually, it is a subset of
the annotation obtained by getAnnotation or
a subset of possibly embedded annotation with the input
counts table. This parameter is optional and required
only when diagplotType is any of "biodetection",
"countsbio", "saturation",
"rnacomp", "readnoise", "biodist",
"gcbias", "lengthbias" or
"filtered".
Regarding contrastList, this parameter is optional
and required only when diagplotType is any of
"deheatmap", "volcano" or "biodist".
It can also be a named structured list of contrasts as
returned by the internal function
metaseqR2:::makeContrastList.
Regarding diagplotType, many of these plots
require the presence of additional package, something
that is checked while running the main metaseqr2 function.
The supported plots are "mds", "biodetection",
"countsbio", "saturation", "rnacomp",
"boxplot", "gcbias", "lengthbias",
"meandiff", "meanvar", "deheatmap",
"volcano", "biodist", "filtered",
"readnoise", "venn", "correl",
"pairwise". For a brief description of these plots
please see the main metaseqr2 help page.
Regarding pList, this parameter is optional
and required only when diagplotType is any of
"deheatmap", "volcano" or "biodist".
Regarding output, supported mechanisms are:
"png", "jpg", "bmp", "pdf",
"ps" or "json". The latter is currently
available for the creation of interactive volcano plots
only when reporting the output, through the highcharts
javascript library. The default plotting ("x11")
is not supported due to instability in certain devices.
A named list containing the file names of the produced
plots. Each list member is names according to the
selected plotting device and is also a named list, whose
names are the plot types. The final contents are the file
names in case the plots are written to a physical
location (not meaningful for "x11").
In order to make the best out of this function, you should generally provide the annotation argument as most and also the most informative plots depend on this. If you don't know what is inside your counts table or how many annotation elements you can provide by embedding it, it's always best to setup a local databse so as to use predefined annotations that work better with the functions of the whole package.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" metaseqrPlot(dataMatrix,sampleList,plotType=c("mds","boxplot")) normArgs <- getDefaults("normalization","deseq2") object <- normalizeDeseq2(dataMatrix,sampleList,normArgs) metaseqrPlot(object,sampleList,plotType="boxplot") ## More #p <- statDeseq2(object,sampleList) #metaseqrPlot(object,sampleList,contrastList=contrast,pList=p, # plotType="volcano")dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" metaseqrPlot(dataMatrix,sampleList,plotType=c("mds","boxplot")) normArgs <- getDefaults("normalization","deseq2") object <- normalizeDeseq2(dataMatrix,sampleList,normArgs) metaseqrPlot(object,sampleList,plotType="boxplot") ## More #p <- statDeseq2(object,sampleList) #metaseqrPlot(object,sampleList,contrastList=contrast,pList=p, # plotType="volcano")
This function calculates the combined p-values when multiple statistical algorithms are applied to the input dataset. It is a helper and it requires very specific arguments so it should not be used individually
metaTest(cpList, metaP = c("simes", "bonferroni", "fisher", "harmonic", "dperm_min", "dperm_max", "dperm_weight", "fperm", "whitlock", "minp", "maxp", "weight", "pandora", "none"), counts, sampleList, statistics, statArgs, libsizeList, nperm = 10000, weight = rep(1/length(statistics), length(statistics)), pOffset = NULL, rc = NULL)metaTest(cpList, metaP = c("simes", "bonferroni", "fisher", "harmonic", "dperm_min", "dperm_max", "dperm_weight", "fperm", "whitlock", "minp", "maxp", "weight", "pandora", "none"), counts, sampleList, statistics, statArgs, libsizeList, nperm = 10000, weight = rep(1/length(statistics), length(statistics)), pOffset = NULL, rc = NULL)
cpList |
a named list whose names are the contrasts
requested from metaseqr2. Each member is a p-value matrix
whose colnames are the names of the statistical tests
applied to the data. See the main |
metaP |
the p-value combination method to use. See
the main |
counts |
the normalized and possibly filtered read
counts matrix. See the main |
sampleList |
the list containing condition names
and the samples under each condition. See the main
|
statistics |
the statistical algorithms used in
metaseqr2. See the main |
statArgs |
the parameters for each statistical
argument. See the main |
libsizeList |
a list with library sizes. See the
main |
nperm |
the number of permutations (Monte Carlo simulations) to perform. |
weight |
a numeric vector of weights for each statistical algorithm. |
pOffset |
|
rc |
the fraction of the available cores to use in a multicore system. |
Ideally one would want to create the same set of indices
for a given dataset so as to create reproducible p-values.
To achieve this, use the set.seed function prior
to any calculations.
A named list with combined p-values. The names are the contrasts and the list members are combined p-value vectors, one for each contrast.
Panagiotis Moulos
cpList <- list(a=matrix(runif(100),50,2)) metaP <- metaTest(cpList,"simes")cpList <- list(a=matrix(runif(100),50,2)) metaP <- metaTest(cpList,"simes")
This data set contains RNA-Seq gene read counts for 3
chromosomes. The data were downloaded from the ENCODE
public repository and are derived from the study of
Mortazavi et al., 2008 (Mortazavi A, Williams BA, McCue
K, Schaeffer L, Wold B. Mapping and quantifying mammalian
transcriptomes by RNA-Seq. Nat Methods. 2008
Jul;5(7):621-8). In their experiment, the authors studied
among others genes expression at two developmental stages
of mouse liver cells. It has two conditions-developmental
stages (e14.5, adult_8_weeks) and four samples (e14.5_1,
e14.5_2, a8w_1, a8w_2). It also contains a predefined
sampleList and libsizeList named
sampleListMm9 and libsizeListMm9.
a data.frame with gene read counts and some embedded
annotation, one row per gene.
Panagiotis Moulos
ENCODE (http://genome.ucsc.edu/encode/)
This function is a wrapper over ABSSeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeAbsseq(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))normalizeAbsseq(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of DESeq normalization
parameters. See the result of
|
output |
the class of the output object. It can be
|
A matrix or a ABSDataSet with normalized counts.
Dionysios Fanidis
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeAbsseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeAbsseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over DESeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeDeseq(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))normalizeDeseq(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of DESeq normalization
parameters. See the result of
|
output |
the class of the output object. It can be
|
A matrix or a CountDataSet with normalized counts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDeseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDeseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over DESeq2 normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeDeseq2(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))normalizeDeseq2(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of DESeq normalization
parameters. See the result of
|
output |
the class of the output object. It can be
|
A matrix or a DESeqDataSet with normalized counts.
Dionysios Fanidis
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDeseq2(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDeseq2(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over ABSSeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeDss(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))normalizeDss(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of DESeq normalization
parameters. See the result of
|
output |
the class of the output object. It can be
|
A matrix or a SeqCountSet with normalized counts.
Dionysios Fanidis
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDss(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeDss(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over EDASeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeEdaseq(geneCounts, sampleList, normArgs = NULL, geneData = NULL, output = c("matrix", "native"))normalizeEdaseq(geneCounts, sampleList, normArgs = NULL, geneData = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of EDASeq normalization
parameters. See the result of
|
geneData |
an optional annotation data frame (such
the ones produced by |
output |
the class of the output object. It can be
|
A matrix or a SeqExpressionSet with normalized counts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix),gc_content=gc, row.names=rownames(dataMatrix) ) normDataMatrix <- normalizeEdaseq(dataMatrix,sampleList, geneData=geneData) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix),gc_content=gc, row.names=rownames(dataMatrix) ) normDataMatrix <- normalizeEdaseq(dataMatrix,sampleList, geneData=geneData) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over edgeR normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeEdger(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))normalizeEdger(geneCounts, sampleList, normArgs = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of edgeR normalization
parameters. See the result of
|
output |
the class of the output object. It can be
|
A matrix or a DGEList with normalized counts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeEdger(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeEdger(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over DESeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeNbpseq(geneCounts, sampleList, normArgs = NULL, libsizeList = NULL, output = c("matrix", "native"))normalizeNbpseq(geneCounts, sampleList, normArgs = NULL, libsizeList = NULL, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of NBPSeq normalization
parameters. See the result of
|
libsizeList |
an optional named list where names
represent samples (MUST be the same as the samples in
|
output |
the class of the output object. It can be
|
A matrix with normalized counts or a list with the normalized counts and other NBPSeq specific parameters.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeNbpseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) normDataMatrix <- normalizeNbpseq(dataMatrix,sampleList) diagplotBoxplot(normDataMatrix,sampleList)
This function is a wrapper over NOISeq normalization. It accepts a matrix of gene counts (e.g. produced by importing an externally generated table of counts to the main metaseqr2 pipeline).
normalizeNoiseq(geneCounts, sampleList, normArgs = NULL, geneData = NULL, logOffset = 1, output = c("matrix", "native"))normalizeNoiseq(geneCounts, sampleList, normArgs = NULL, geneData = NULL, logOffset = 1, output = c("matrix", "native"))
geneCounts |
a table where each row represents a gene and each column a sample. Each cell contains the read counts for each gene and sample. Such a table can be produced outside metaseqr2 and is imported during the basic metaseqr2 workflow. |
sampleList |
the list containing condition names and the samples under each condition. |
normArgs |
a list of NOISeq normalization
parameters. See the result of
|
geneData |
an optional annotation data frame (such
the ones produced by |
logOffset |
an offset to use to avoid infinity in logarithmic data transformations. |
output |
the class of the output object. It can be
|
A matrix with normalized counts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix),gc_content=gc, biotype=rep("protein_coding",nrow(dataMatrix)), row.names=rownames(dataMatrix) ) normDataMatrix <- normalizeNoiseq(dataMatrix,sampleList,normArgs=NULL,geneData) diagplotBoxplot(normDataMatrix,sampleList)dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) diagplotBoxplot(dataMatrix,sampleList) lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix),gc_content=gc, biotype=rep("protein_coding",nrow(dataMatrix)), row.names=rownames(dataMatrix) ) normDataMatrix <- normalizeNoiseq(dataMatrix,sampleList,normArgs=NULL,geneData) diagplotBoxplot(normDataMatrix,sampleList)
This function is a helper for the metaseqr2
pipeline, for reading SAM/BAM or BED files when a read
counts file is not available. It can also be used
very easily in an autonomous manner.
read2count(targets, annotation, fileType = targets$type, transLevel = "gene", utrOpts = list(frac = 1, minLength = 300, downstream = 50), interFeature = FALSE, rc = NULL)read2count(targets, annotation, fileType = targets$type, transLevel = "gene", utrOpts = list(frac = 1, minLength = 300, downstream = 50), interFeature = FALSE, rc = NULL)
targets |
a named list, the output of
|
annotation |
a |
fileType |
the type of raw input files. It can be
|
transLevel |
see the |
utrOpts |
a named list with members |
interFeature |
see the |
rc |
the fraction of the available cores to use in a multicore system. |
A data frame with counts for each sample, ready to be
passed to the main metaseqr2 pipeline.
Panagiotis Moulos
dataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) geneData <- loadAnnotation("mm10","ensembl","gene") myTargets <- readTargets(file.path(path,"targets.txt")) if (.Platform$OS.type == "unix") { r2c <- read2count(targets=myTargets, fileType=myTargets$type,annotation=geneData) geneCounts <- r2c$counts libsizeList <- r2c$libsize }dataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) geneData <- loadAnnotation("mm10","ensembl","gene") myTargets <- readTargets(file.path(path,"targets.txt")) if (.Platform$OS.type == "unix") { r2c <- read2count(targets=myTargets, fileType=myTargets$type,annotation=geneData) geneCounts <- r2c$counts libsizeList <- r2c$libsize }
Create the main sample list and determine the BAM/BED files for each sample from an external file.
readTargets(input, path = NULL)readTargets(input, path = NULL)
input |
a tab-delimited file or a YAML file specifically structured. See Details. |
path |
an optional path where all the BED/BAM files are placed, to be prepended to the BAM/BED file names in the targets file. |
Regarding the input file, this can be a simple text tab-delimited file or a YAML file describing the data to be analyzed.
Regarding the tab-delimited version, its columns must
be structured as follows: the first line of the external
tab delimited file should contain column names (names
are not important). The first column MUST contain UNIQUE
sample names. The second column MUST contain the raw
BAM/BED files WITH their full path. Alternatively, the
path argument should be provided (see below). The
third column MUST contain the biological condition where
each of the samples in the first column should belong to.
There is an optional fourth column which should contain
the keywords "single" for single-end reads,
"paired" for paired-end reads or "mixed"
for BAM files that contain both single- and paired-end
reads (e.g. after a mapping procedure with two round of
alignment). If this column is not provided, single-end
reads will be assumed. There is an optional fifth column
which stranded read assignment. It should contain the
keywords "forward" for a forward (5'->3') strand
library construction protocol, "reverse" for a
reverse (3'->5') strand library construction protocol,
or "no" for unstranded/unknown protocol. If this
column is not provided, unstranded reads will be assumed.
Regarding the YAML version, the same instructions apply,
but this time instead of columns, the data are provided
as a YAML array under a keyword/top-level field
representing the respective header in the tab-delimited
version. Alternatively, the aforementioned structure
can be nested under a root level named strictly either
targets or metaseqR2_targets. The latter
can be especially useful when incorporating the
metaseqR2 pipeline in a wider pipeline including various
analyses and described using a workflow language such
as CWL.
A named list with four members. The first member is
a named list whose names are the conditions of the
experiments and its members are the samples belonging
to each condition. The second member is like the
first, but this time the members are named vectors
whose names are the sample names and the vector
elements are full path to BAM/BED files. The third
member is like the second, but instead of filenames
it contains information about single- or paired-end
reads (if available). The fourth member is like the
second, but instead of filenames it contains
information about the strandedness of the reads (if
available). The fifth member is the guessed type
of the input files (SAM/BAM or BED). It will be used
if not given in the main read2count
function.
Panagiotis Moulos
dataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() # Tab delimited case write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) theList <- readTargets(file.path(path,"targets.txt"),path=path) sampleList <- theList$samples bamfileList <- theList$files # YAML case require(yaml) write_yaml(as.list(targets),file.path(path,"targets.yml")) theYList <- readTargets(file.path(path,"targets.yml"),path=path) identical(theList,theYList) # TRUE # YAML case with nested targets write_yaml(list(targets=as.list(targets)), file.path(path,"targets2.yml")) theYList2 <- readTargets(file.path(path,"targets2.yml"),path=path) identical(theYList,theYList2) # TRUEdataPath <- system.file("extdata",package="metaseqR2") targets <- data.frame(samplename=c("C","T"), filename=file.path(dataPath,c("C.bam","T.bam")), condition=c("Control","Treatment"), paired=c("single","single"),stranded=c("forward","forward")) path <- tempdir() # Tab delimited case write.table(targets,file=file.path(path,"targets.txt"), sep="\t",row.names=FALSE,quote=FALSE) theList <- readTargets(file.path(path,"targets.txt"),path=path) sampleList <- theList$samples bamfileList <- theList$files # YAML case require(yaml) write_yaml(as.list(targets),file.path(path,"targets.yml")) theYList <- readTargets(file.path(path,"targets.yml"),path=path) identical(theList,theYList) # TRUE # YAML case with nested targets write_yaml(list(targets=as.list(targets)), file.path(path,"targets2.yml")) theYList2 <- readTargets(file.path(path,"targets2.yml"),path=path) identical(theYList,theYList2) # TRUE
The sample list for mm9GeneCounts. See the data
set description.
a named list with condition and sample names.
Panagiotis Moulos
ENCODE (http://genome.ucsc.edu/encode/)
This function is a wrapper over DESeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statAbsseq(object, sampleList, contrastList = NULL, statArgs = NULL)statAbsseq(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of DESeq statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Dionysios Fanidis
require(ABSSeq) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeAbsseq(dataMatrix,sampleList) p <- statAbsseq(normDataMatrix,sampleList,contrast)require(ABSSeq) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeAbsseq(dataMatrix,sampleList) p <- statAbsseq(normDataMatrix,sampleList,contrast)
This function is a wrapper over DESeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statDeseq(object, sampleList, contrastList = NULL, statArgs = NULL)statDeseq(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of DESeq statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- metaseqR2:::newCountDataSet(dataMatrix, c("A","A","B","B","B")) normDataMatrix <- normalizeDeseq(dataMatrix,sampleList) p <- statDeseq(normDataMatrix,sampleList,contrast)dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- metaseqR2:::newCountDataSet(dataMatrix, c("A","A","B","B","B")) normDataMatrix <- normalizeDeseq(dataMatrix,sampleList) p <- statDeseq(normDataMatrix,sampleList,contrast)
This function is a wrapper over DESeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statDeseq2(object, sampleList, contrastList = NULL, statArgs = NULL)statDeseq2(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of DESeq statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Dionysios Fanidis
require(DESeq2) dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeDeseq2(dataMatrix,sampleList) p <- statDeseq2(normDataMatrix,sampleList,contrast)require(DESeq2) dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeDeseq2(dataMatrix,sampleList) p <- statDeseq2(normDataMatrix,sampleList,contrast)
This function is a wrapper over DESeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statDss(object, sampleList, contrastList = NULL, statArgs = NULL)statDss(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of DESeq statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Dionysios Fanidis
require(DSS) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeDss(dataMatrix,sampleList) p <- statDss(normDataMatrix,sampleList,contrast)require(DSS) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeDss(dataMatrix,sampleList) p <- statDss(normDataMatrix,sampleList,contrast)
This function is a wrapper over edgeR statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statEdger(object, sampleList, contrastList = NULL, statArgs = NULL)statEdger(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of edgeR statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Panagiotis Moulos
require(edgeR) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(normDataMatrix,sampleList,contrast)require(edgeR) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeEdger(dataMatrix,sampleList) p <- statEdger(normDataMatrix,sampleList,contrast)
This function is a wrapper over limma statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statLimma(object, sampleList, contrastList = NULL, statArgs = NULL)statLimma(object, sampleList, contrastList = NULL, statArgs = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of edgeR statistical algorithm
parameters. See the result of
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
Panagiotis Moulos
require(limma) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeEdger(dataMatrix,sampleList) p <- statLimma(normDataMatrix,sampleList,contrast)require(limma) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeEdger(dataMatrix,sampleList) p <- statLimma(normDataMatrix,sampleList,contrast)
This function is a wrapper over NBPSeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statNbpseq(object, sampleList, contrastList = NULL, statArgs = NULL, libsizeList = NULL)statNbpseq(object, sampleList, contrastList = NULL, statArgs = NULL, libsizeList = NULL)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of NBPSeq statistical algorithm
parameters. See the result of
|
libsizeList |
an optional named list where names
represent samples (MUST be the same as the samples
|
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of p-values, whose names are the names of the contrasts.
There is currently a problem with the NBPSeq package and the workflow that is specific to the NBPSeq package. The problem has to do with function exporting as there are certain functions which are not recognized from the package internally. For this reason and until it is fixed, only the Smyth workflow will be available with the NBPSeq package.
Panagiotis Moulos
require(NBPSeq) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeNbpseq(dataMatrix,sampleList) p <- statNbpseq(normDataMatrix,sampleList,contrast)require(NBPSeq) dataMatrix <- metaseqR2:::exampleCountData(2000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" normDataMatrix <- normalizeNbpseq(dataMatrix,sampleList) p <- statNbpseq(normDataMatrix,sampleList,contrast)
This function is a wrapper over NOISeq statistical testing. It accepts a matrix of normalized gene counts or an S4 object specific to each normalization algorithm supported by metaseqR2.
statNoiseq(object, sampleList, contrastList = NULL, statArgs = NULL, geneData = NULL, logOffset = 1)statNoiseq(object, sampleList, contrastList = NULL, statArgs = NULL, geneData = NULL, logOffset = 1)
object |
a matrix or an object specific to each normalization algorithm supported by metaseqR2, containing normalized counts. See also Details. |
sampleList |
the list containing condition names and the samples under each condition. |
contrastList |
vector of contrasts as defined in the
main help page of |
statArgs |
a list of edgeR statistical algorithm
parameters. See the result of
|
geneData |
an optional annotation data frame (such
the ones produced by |
logOffset |
a number to be added to each element of data matrix in order to avoid Infinity on log type data transformations. |
Regarding object, apart from matrix (also
for NOISeq), the object can be a SeqExpressionSet
(EDASeq), CountDataSet (DESeq), DGEList
(edgeR), DESeqDataSet (DESeq2), SeqCountSet
(DSS) or ABSDataSet (ABSSeq).
Regarding contrastList it can also be a named
structured list of contrasts as returned by the internal
function metaseqR2:::makeContrastList.
A named list of NOISeq q-values, whose names are the names of the contrasts.
Panagiotis Moulos
dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) biotype=rep("protein_coding",nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix), gc_content=gc,biotype=biotype ) normArgs <- metaseqR2:::getDefaults("normalization","noiseq") normDataMatrix <- normalizeNoiseq(dataMatrix,sampleList,normArgs, geneData) p <- statNoiseq(normDataMatrix,sampleList,contrast, geneData=geneData)dataMatrix <- metaseqR2:::exampleCountData(1000) sampleList <- list(A=c("A1","A2"),B=c("B1","B2","B3")) contrast <- "A_vs_B" lengths <- round(1000*runif(nrow(dataMatrix))) starts <- round(1000*runif(nrow(dataMatrix))) ends <- starts + lengths gc=runif(nrow(dataMatrix)) biotype=rep("protein_coding",nrow(dataMatrix)) geneData <- data.frame( chromosome=c(rep("chr1",nrow(dataMatrix)/2), rep("chr2",nrow(dataMatrix)/2)), start=starts,end=ends,gene_id=rownames(dataMatrix), gc_content=gc,biotype=biotype ) normArgs <- metaseqR2:::getDefaults("normalization","noiseq") normDataMatrix <- normalizeNoiseq(dataMatrix,sampleList,normArgs, geneData) p <- statNoiseq(normDataMatrix,sampleList,contrast, geneData=geneData)