| Title: | Statistical Analysis of MPRA data |
|---|---|
| Description: | MPRAnalyze provides statistical framework for the analysis of data generated by Massively Parallel Reporter Assays (MPRAs), used to directly measure enhancer activity. MPRAnalyze can be used for quantification of enhancer activity, classification of active enhancers and comparative analyses of enhancer activity between conditions. MPRAnalyze construct a nested pair of generalized linear models (GLMs) to relate the DNA and RNA observations, easily adjustable to various experimental designs and conditions, and provides a set of rigorous statistical testig schemes. |
| Authors: | Tal Ashuach [aut, cre], David S Fischer [aut], Anat Kriemer [ctb], Fabian J Theis [ctb], Nir Yosef [ctb], |
| Maintainer: | Tal Ashuach <[email protected]> |
| License: | GPL-3 |
| Version: | 1.25.0 |
| Built: | 2024-10-30 08:21:30 UTC |
| Source: | https://github.com/bioc/MPRAnalyze |
Run a comparative analysis between conditions
analyzeComparative( obj, rnaDesign, dnaDesign = NULL, fit.se = FALSE, reducedDesign = NULL, correctControls = TRUE, verbose = TRUE, mode = "classic", BPPARAM = NULL )analyzeComparative( obj, rnaDesign, dnaDesign = NULL, fit.se = FALSE, reducedDesign = NULL, correctControls = TRUE, verbose = TRUE, mode = "classic", BPPARAM = NULL )
obj |
the MpraObject |
rnaDesign |
the design for the RNA model. |
dnaDesign |
the design for the DNA model. Only terms that are matched with the RNA design should be included. |
fit.se |
logical, if TRUE the standard errors of the coefficients are extracted from the model. These are necessary for computing coefficient- based testing, but make the model fitting slower. Deafult: FALSE |
reducedDesign |
the design for the reduced RNA model, for a likelihood- ratio testing scheme. The Reduced design must be nested within the full design (i.e all terms in the reduced must be included in the full). |
correctControls |
if TRUE (default), use the negative controls to establish the null hypothesis, correcting for systemic bias in the data |
verbose |
print progress reports (default: TRUE) |
mode |
whether to run in classic mode ("classic") or in scalable mode ("scale"). Scale mode is only available in situations when each RNA observation has a single corresponding DNA observation. |
BPPARAM |
a parallelization object created by BiocParallel. This overwrites the BPPARAM object set in the object creation. |
the MpraObject with fitted models for the input enhancers
data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## run an LRT-based analysis, as recommnded: obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) ## alternatively, run a coefficient-based analysis: obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, fit.se = TRUE)data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## run an LRT-based analysis, as recommnded: obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) ## alternatively, run a coefficient-based analysis: obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, fit.se = TRUE)
epirical: the model is fitted as specified, enabling future empirical testing (either empirical p-value if negative controls are provided, or a global devience analysis, see details in 'test.empirical')
lrt: only available if negative controls are provided. A likelihood ratio test is used, with the null hypothesis a joint model of the controls and a given candidate sequence, and the alternative model being a separate model for controls and candidates.
analyzeQuantification(obj, dnaDesign = ~1, rnaDesign = ~1, BPPARAM = NULL)analyzeQuantification(obj, dnaDesign = ~1, rnaDesign = ~1, BPPARAM = NULL)
obj |
the MpraObject |
dnaDesign |
the design of the DNA counts |
rnaDesign |
the design of the RNA counts |
BPPARAM |
a parallelization object created by BiocParallel. This overwrites the BPPARAM object set in the object creation. |
the MpraObject, with populated models
data <- simulateMPRA(tr = rep(2,10), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1)data <- simulateMPRA(tr = rep(2,10), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1)
A subset of MPRA data from Inoue et al., comparing enhancer activity of episomal constructs vs. chromosomally integrated constructs (integration was performed with a lentivirus). Data included negative control enhancers, multiple batches and barcodes, a subsample of which are included in this sample data for runtime purposes.
data(ChrEpi) ce.colAnnot ce.dnaCounts ce.rnaCounts ce.controldata(ChrEpi) ce.colAnnot ce.dnaCounts ce.rnaCounts ce.control
Column annotations for each column (sample) in the data matrices
batch identifier, factor
condition identifier, factor. WT corresponds to chromosomal and MT corresponds to episomal
barcode identifier, factor
DNA observations
DNA observations
indices of control enhancers
An object of class data.frame with 40 rows and 3 columns.
An object of class matrix with 110 rows and 40 columns.
An object of class matrix with 110 rows and 40 columns.
An object of class logical of length 110.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5204343/
estimate library size correction factors
estimateDepthFactors( obj, lib.factor = NULL, which.lib = "both", depth.estimator = "uq" )estimateDepthFactors( obj, lib.factor = NULL, which.lib = "both", depth.estimator = "uq" )
obj |
the MpraObject |
lib.factor |
the factor associating each sample to a library. Can be a factor or the name of a column in the object's colAnnot. If not provided, the data is assumed to have been generated from a single library, and constant library depth is set. |
which.lib |
which library to compute the depth factors for. Options are "both" (default), "dna" or "rna". If the DNA and RNA counts have different library factors, this function should be called twice: once with "dna" and once with "rna" |
depth.estimator |
a character indicating which depth estimation to use, or a function to perform the estimation. Currently supported values are "uq" for upper quantile of non-zero values (default), "rle" for RLE (uses geometric mean, and is therefore not recommended if libraries have 0 counts), or "totsum" for total sum. For a function input: function should take a numeric vector and return a single numeric, and preferably handle NA values. See examples. |
the MpraObject with estimated values for sequencing depth factors
since in most MPRA experiments multiple barcodes exist within a single library, each column in the matrix is usually not a separate library. For this reason, it is recommended to supply this function with the appropriate partitioning of the data matrix columns into libraries, see lib.factor
data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## Upper quantile, using a higher quantile than 0.75: obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both", depth.estimator = function(x) quantile(x, .95, na.rm=TRUE))data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## Upper quantile, using a higher quantile than 0.75: obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both", depth.estimator = function(x) quantile(x, .95, na.rm=TRUE))
return the fitted value for the transcription rate.
getAlpha(obj, by.factor = NULL, full = TRUE)getAlpha(obj, by.factor = NULL, full = TRUE)
obj |
the MpraObject to extract from, must be after model fitting |
by.factor |
return a matrix of values, corresponding to the estimated rates of transcription under different values of a factor included in the design. Value must be of these options: NULL: (default) return only the intercept term, a single baseline rate for each enhancer "all": will return the corresponding transcription rates for all values included in the model factor name: must be a factor included in the RNA annotations and the rna design. Will return the corresponding rates for all values of the given factor |
full |
if true, return rate of the full model (default), otherwise of the reduced model (only applies if an LRT-based analysis was used) |
the estimate for transcription rate as fitted by the model
data <- simulateMPRA(tr = rep(2,10), da=c(rep(0,5), rep(1,5)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) ## get alpha estimate for the two conditions alpha <- getAlpha(obj, by.factor="condition")data <- simulateMPRA(tr = rep(2,10), da=c(rep(0,5), rep(1,5)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) ## get alpha estimate for the two conditions alpha <- getAlpha(obj, by.factor="condition")
Get model distribution parameters from an MpraObject of a given candidate enhancer
getDistrParam_DNA(obj, enhancer, full = TRUE) getDistrParam_RNA(obj, enhancer = NULL, full = TRUE)getDistrParam_DNA(obj, enhancer, full = TRUE) getDistrParam_RNA(obj, enhancer = NULL, full = TRUE)
obj |
MpraObject to extract from |
enhancer |
enhancer to extract |
full |
whether to extract from full model |
fit parameters (numeric, samples x parameters)
data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) ## get distributional parameters of the first enhancer: dist.params.dna <- getDistrParam_DNA(obj, 1) dist.params.rna <- getDistrParam_RNA(obj, 1)data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) ## get distributional parameters of the first enhancer: dist.params.dna <- getDistrParam_DNA(obj, 1) dist.params.rna <- getDistrParam_RNA(obj, 1)
Get DNA model-based estimates from an MpraObject (the expected values based on the model). These can be compared with the observed counts to assess goodness of fit.
getFits_DNA( obj, enhancers = NULL, depth = TRUE, full = TRUE, transition = FALSE )getFits_DNA( obj, enhancers = NULL, depth = TRUE, full = TRUE, transition = FALSE )
obj |
MpraObject to extract from |
enhancers |
which enhancers to get the fits for. Can be character vectors with enhancer names, logical or numeric enhancer indices, or NULL if all enhancers are to be extracted (default) |
depth |
include depth correction in the model fitting (default TRUE) |
full |
if LRT modeling was used, TRUE (default) would return the fits of the full model, FALSEwould return the reduced model fits. |
transition |
use the DNA->RNA transition matrix (deafult: FALSE). This is useful if the DNA observations need to be distributed to match the RNA observations. |
DNA fits (numeric, enhancers x samples)
data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) dna.fits <- getFits_DNA(obj)data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) dna.fits <- getFits_DNA(obj)
Get RNA model-based estimates from an MpraObject (the expected values based on the model). These can be compared with the observed counts to assess goodness of fit.
getFits_RNA(obj, enhancers = NULL, depth = TRUE, full = TRUE, rnascale = TRUE)getFits_RNA(obj, enhancers = NULL, depth = TRUE, full = TRUE, rnascale = TRUE)
obj |
MpraObject to extract from |
enhancers |
which enhancers to get the fits for. Can be character vectors with enhancer names, logical or numeric enhancer indices, or NULL if all enhancers are to be extracted (default) |
depth |
include depth correction in the model fitting (default TRUE) |
full |
if LRT modeling was used, TRUE (default) would return the fits of the full model, FALSEwould return the reduced model fits. |
rnascale |
if controls were used to correct the fitting (in comparative analyses), use these factors to re-adjust the estimates back. |
RNA fits (numeric, enhancers x samples)
data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) rna.fits <- getFits_RNA(obj)data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) rna.fits <- getFits_RNA(obj)
extract the DNA model parameters
getModelParameters_DNA(obj, features = NULL, full = TRUE) getModelParameters_RNA(obj, features = NULL, full = TRUE)getModelParameters_DNA(obj, features = NULL, full = TRUE) getModelParameters_RNA(obj, features = NULL, full = TRUE)
obj |
the MpraObject to extract the parameters from |
features |
the features to extract the parameters from (by default, parameters will be returned for all features) |
full |
if TRUE (default), return the parameters of the full model. Otherwise, return the parameters of the reduced model (only relevant for LRT-based analyses) |
a data.frame of features (rows) by parameters (cols). By convension, the first parameter is related to the second moment, and the interpretation of it depends on the distributional model used ('alpha' for 'gamma.pois', variance for 'ln.nb' and 'ln.ln')
data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) model.params.dna <- getModelParameters_DNA(obj) model.params.rna <- getModelParameters_RNA(obj)data <- simulateMPRA(tr = rep(2,5), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) model.params.dna <- getModelParameters_DNA(obj) model.params.rna <- getModelParameters_RNA(obj)
The main object MPRAnalyze works with, contains the input data, associated annotations, model parameters and analysis results.
MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA_integer_, rowAnnot = NULL, BPPARAM = NULL ) ## S4 method for signature 'matrix' MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA_integer_, rowAnnot = NULL, BPPARAM = NULL ) ## S4 method for signature 'SummarizedExperiment' MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA, rowAnnot = NULL, BPPARAM = NULL ) dnaCounts(obj) ## S4 method for signature 'MpraObject' dnaCounts(obj) rnaCounts(obj) ## S4 method for signature 'MpraObject' rnaCounts(obj) dnaAnnot(obj) ## S4 method for signature 'MpraObject' dnaAnnot(obj) rnaAnnot(obj) ## S4 method for signature 'MpraObject' rnaAnnot(obj) rowAnnot(obj) ## S4 method for signature 'MpraObject' rowAnnot(obj) controls(obj) ## S4 method for signature 'MpraObject' controls(obj) dnaDepth(obj) ## S4 method for signature 'MpraObject' dnaDepth(obj) rnaDepth(obj) ## S4 method for signature 'MpraObject' rnaDepth(obj) model(obj) ## S4 method for signature 'MpraObject' model(obj)MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA_integer_, rowAnnot = NULL, BPPARAM = NULL ) ## S4 method for signature 'matrix' MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA_integer_, rowAnnot = NULL, BPPARAM = NULL ) ## S4 method for signature 'SummarizedExperiment' MpraObject( dnaCounts, rnaCounts, dnaAnnot = NULL, rnaAnnot = NULL, colAnnot = NULL, controls = NA, rowAnnot = NULL, BPPARAM = NULL ) dnaCounts(obj) ## S4 method for signature 'MpraObject' dnaCounts(obj) rnaCounts(obj) ## S4 method for signature 'MpraObject' rnaCounts(obj) dnaAnnot(obj) ## S4 method for signature 'MpraObject' dnaAnnot(obj) rnaAnnot(obj) ## S4 method for signature 'MpraObject' rnaAnnot(obj) rowAnnot(obj) ## S4 method for signature 'MpraObject' rowAnnot(obj) controls(obj) ## S4 method for signature 'MpraObject' controls(obj) dnaDepth(obj) ## S4 method for signature 'MpraObject' dnaDepth(obj) rnaDepth(obj) ## S4 method for signature 'MpraObject' rnaDepth(obj) model(obj) ## S4 method for signature 'MpraObject' model(obj)
dnaCounts |
the DNA count matrix, or a SummarizedExperiment object containing the DNA Counts and column annotations for the DNA data. If the input is a SummarizedExperiment object, the dnaAnnot (or colAnnot) arguments will be ignored |
rnaCounts |
the RNA count matrix, or a SummarizedExperiment object containing the RNA Counts and column annotations for the RNA data. If the input is a SummarizedExperiment object, the rnaAnnot (or colAnnot) arguments will be ignored |
dnaAnnot |
data.frame with the DNA column (sample) annotations |
rnaAnnot |
data.frame with the RNA column (sample) annotations |
colAnnot |
if annotations for DNA and RNA are identical, they can be set at the same time using colAnnot instead of using both rnaAnnot and dnaAnnot |
controls |
IDs of the rows in the matrices that correspond to negative control enhancers. These are used to establish the null for quantification purposes, and to correct systemic bias in comparative analyses. Can be a character vectors (corresponding to rownames in the data matrices), logical or numeric indices. |
rowAnnot |
a data.frame with the row (candidate enhancer) annotations. The names must match the row names in the DNA and RNA count matrices. |
BPPARAM |
a parallelization backend using the BiocParallel package, see more details [here](http://bioconductor.org/packages/release/bioc/html/BiocParallel.html) |
obj |
The MpraObject to extract properties from |
an initialized MpraObject
MpraObject properties can be accessed using accessor functions
the DNA count matrix
the RNA count matrix
data.frame with the DNA column (sample) annotations
data.frame with the RNA column (sample) annotations
data.frame with the row (candidate enhancers) annotations
the distributional model used. the Gamma-Poisson convolutional
model is used by default. see setModel
The library size correction factors computed for the DNA
libraries. These are computed by the estimateDepthFactors
function and can be set manually using the setDepthFactors
function
The library size correction factors computed for the RNA
libraries These are computed by the estimateDepthFactors
function and can be set manually using the setDepthFactors
function
data <- simulateMPRA(tr = rep(2,10), da=c(rep(2,5), rep(2.5,5)), nbatch=2, nbc=20) ## use 3 of the non-active enhancers as controls obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot, controls = as.integer(c(1,2,4))) ## alternatively, initialize the object with SummarizedExperiment objects: ## Not run: se.DNA <- SummarizedExperiment(list(data$obs.dna), colData=data$annot) se.RNA <- SummarizedExperiment(list(data$obs.rna), colData=data$annot) obj <- MpraObject(dnaCounts = se.DNA, rnaCounts = rna.se, controls = as.integer(c(1,2,4))) ## End(Not run) dnaCounts <- dnaCounts(obj) rnaCounts <- rnaCounts(obj) dnaAnnot <- dnaAnnot(obj) rnaAnnot <- rnaAnnot(obj) controls <- controls(obj) rowAnnot <- rowAnnot(obj) model <- model(obj) obj <- estimateDepthFactors(obj, lib.factor=c("batch", "condition")) dnaDepth <- dnaDepth(obj) rnaDepth <- rnaDepth(obj)data <- simulateMPRA(tr = rep(2,10), da=c(rep(2,5), rep(2.5,5)), nbatch=2, nbc=20) ## use 3 of the non-active enhancers as controls obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot, controls = as.integer(c(1,2,4))) ## alternatively, initialize the object with SummarizedExperiment objects: ## Not run: se.DNA <- SummarizedExperiment(list(data$obs.dna), colData=data$annot) se.RNA <- SummarizedExperiment(list(data$obs.rna), colData=data$annot) obj <- MpraObject(dnaCounts = se.DNA, rnaCounts = rna.se, controls = as.integer(c(1,2,4))) ## End(Not run) dnaCounts <- dnaCounts(obj) rnaCounts <- rnaCounts(obj) dnaAnnot <- dnaAnnot(obj) rnaAnnot <- rnaAnnot(obj) controls <- controls(obj) rowAnnot <- rowAnnot(obj) model <- model(obj) obj <- estimateDepthFactors(obj, lib.factor=c("batch", "condition")) dnaDepth <- dnaDepth(obj) rnaDepth <- rnaDepth(obj)
Manually set library depth correction factors
setDepthFactors(obj, dnaDepth, rnaDepth)setDepthFactors(obj, dnaDepth, rnaDepth)
obj |
the MpraObject |
dnaDepth |
library size factors for the DNA data, a numeric vector of length of the number of columns in the DNA data matrix |
rnaDepth |
library size factors for the RNA data, a numeric vector of length of the number of columns in the RNA data matrix |
the MpraObject with library depth factors
data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) ## set constant depth factors (no depth correction) obj <- setDepthFactors(obj, dnaDepth = rep(1, NCOL(data$obs.dna)), rnaDepth = rep(1, NCOL(data$obs.rna)))data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) ## set constant depth factors (no depth correction) obj <- setDepthFactors(obj, dnaDepth = rep(1, NCOL(data$obs.dna)), rnaDepth = rep(1, NCOL(data$obs.rna)))
Set the distributional model used. Default is gamma.pois, and is recommended. Other supoprted models are ln.nb in which the DNA follows a log-normal distribution and the RNA follows a negative binomial, and ln.ln in which both follow log-normal distributions. To use alternative distributional models, use this function before fitting the model.
setModel(obj, model)setModel(obj, model)
obj |
the MPRAnalyze object |
model |
the charater identifier of the model to be used. Currently supported models: "ln.nb", "gamma.pois", "ln.ln" |
the MPRAnalyze with the model set for the given value
data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- setModel(obj, "ln.ln") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1)data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=20) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- setModel(obj, "ln.ln") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1)
Simulate an MPRA dataset
simulateMPRA( tr = rep(2, 100), da = NULL, dna.noise.sd = 0.2, rna.noise.sd = 0.3, dna.inter = 5, dna.inter.sd = 0.5, nbc = 100, coef.bc.sd = 0.5, nbatch = 3, coef.batch.sd = 0.5 )simulateMPRA( tr = rep(2, 100), da = NULL, dna.noise.sd = 0.2, rna.noise.sd = 0.3, dna.inter = 5, dna.inter.sd = 0.5, nbc = 100, coef.bc.sd = 0.5, nbatch = 3, coef.batch.sd = 0.5 )
tr |
a vector of the true transcription rates, in log scale. The length of the vector determines the number of enhancers included in the dataset. Default is 100 enhancers of identical transcription rate of 2. |
da |
a vector determinig differential activity. Values are assumed to be in log scale, and will be used in the model as log Fold-Change values. If NULL (default) a single condition is simulated. |
dna.noise.sd |
level of noise to add to the DNA library |
rna.noise.sd |
level of noise to add to the RNA library |
dna.inter |
the baseline DNA levels (intercept term), controlling the true mean abundance of plasmids |
dna.inter.sd |
the true variation of the plasmid levels |
nbc |
number of unique barcode to include per enhancer |
coef.bc.sd |
true variation between barcodes |
nbatch |
number of batches to simulate |
coef.batch.sd |
the level of true variation that distinguishes batches (the size of the batch effects) |
the data is generated by using the same nested-GLM construct that MPRAnalyzes uses, with non-strandard log-normal noise models (whereas by default MPRAnalyze uses a Gamma-Poisson model). The data generated can have multiple batches, and either 1 or 2 conditions, and the simulated data is always paired (DNA and RNA extracted from the same library). User can control both true and observed variation levels (noise), the number of expected plasmids per barcode, the true transcription ratio, the size of the batch and barcode effects.
a list:
true.dna The true dna abundances
obs.dna the observed dna counts
true.rna the true rna abundances
obs.rna the observed rna counts
annot the annotations data.frame for each sample
# single condition data <- simulateMPRA() # two conditions data <- simulateMPRA(da=c(rep(-0.5, 50), rep(0.5, 50))) # more observed noise data <- simulateMPRA(dna.noise.sd = 0.75, rna.noise.sd = 0.75) # gradually increasing dataset data <- simulateMPRA(tr = seq(2,3,0.01), da=NULL)# single condition data <- simulateMPRA() # two conditions data <- simulateMPRA(da=c(rep(-0.5, 50), rep(0.5, 50))) # more observed noise data <- simulateMPRA(dna.noise.sd = 0.75, rna.noise.sd = 0.75) # gradually increasing dataset data <- simulateMPRA(tr = seq(2,3,0.01), da=NULL)
Calculate the significance of a factor in the regression model
testCoefficient(obj, factor, contrast)testCoefficient(obj, factor, contrast)
obj |
the MpraObject |
factor |
the name of the factor to make the comparison on |
contrast |
the character value of the factor to use as a contrast. See details. |
a data.frame of the results this include the test statistic, logFC, p-value and BH-corrected FDR.
data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## fit.se must be TRUE for coefficient based testing to work obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, fit.se = TRUE) results <- testCoefficient(obj, "condition", "contrast")data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") ## fit.se must be TRUE for coefficient based testing to work obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, fit.se = TRUE) results <- testCoefficient(obj, "condition", "contrast")
test for significant activity (quantitative analysis) using various empirical tests (see details)
testEmpirical( obj, statistic = NULL, useControls = TRUE, twoSided = FALSE, subset = NULL )testEmpirical( obj, statistic = NULL, useControls = TRUE, twoSided = FALSE, subset = NULL )
obj |
the MpraObject, after running an analysis function |
statistic |
if null [default], the intercept term is used as the score. An alternate score can be provided by setting 'statistic'. Must be a numeric vector. |
useControls |
is TRUE and controls are available, use the controls to establish the background model and compare against. This allows for more accurate zscores as well as empircal p-values. |
twoSided |
should the p-value be from a two-sided test (default: FALSE, right-side test) |
subset |
only test a subset of the enhancers in the object (logical, indices or names). Default is NULL, then all the enhancers are included. |
a data.frame of empirical summary statistics based on the model's estimate of slope, or the given statistic. These are:
statistic: the statistic (either the provided, or extracted from the models)
zscore: Z-score of the statistic (number of standard devisations from the mean). If controls are available, the score is based on their distribution: so it's the number of control-sd from the control-mean
mad.score: a median-baed equivalent of the Z-score, with less sensitivity to outlier values. If controls are provided, it's based on their distribution.
pval.zscore: a p-value based on the normal approximation of the Z-scores
pval.empirical: only available if negative controls are provided. empirical P-value, using the control distribution as the null
data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) results <- testEmpirical(obj) ## or test with a different statistic: aggregated.ratio <- rowSums(data$obs.rna) / rowSums(data$obs.dna) results <- testEmpirical(obj, aggregated.ratio)data <- simulateMPRA(tr = rep(2,10), da=NULL, nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeQuantification(obj, dnaDesign = ~ batch + barcode, rnaDesign = ~1) results <- testEmpirical(obj) ## or test with a different statistic: aggregated.ratio <- rowSums(data$obs.rna) / rowSums(data$obs.dna) results <- testEmpirical(obj, aggregated.ratio)
Calculate likelihood ratio test for the specific nested model
testLrt(obj)testLrt(obj)
obj |
the MpraObject containing the full and reduced |
results data frame
Must be run after running an LRT-based analysis
data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) results <- testLrt(obj)data <- simulateMPRA(tr = rep(2,5), da=c(rep(0,2), rep(1,3)), nbatch=2, nbc=15) obj <- MpraObject(dnaCounts = data$obs.dna, rnaCounts = data$obs.rna, colAnnot = data$annot) obj <- estimateDepthFactors(obj, lib.factor = "batch", which.lib = "both") obj <- analyzeComparative(obj, dnaDesign = ~ batch + barcode + condition, rnaDesign = ~ condition, reducedDesign = ~ 1) results <- testLrt(obj)