| Title: | Multi-Dataset Model-based Differential Expression Proteomics Analysis Platform |
|---|---|
| Description: | ProteoMM is a statistical method to perform model-based peptide-level differential expression analysis of single or multiple datasets. For multiple datasets ProteoMM produces a single fold change and p-value for each protein across multiple datasets. ProteoMM provides functionality for normalization, missing value imputation and differential expression. Model-based peptide-level imputation and differential expression analysis component of package follows the analysis described in “A statistical framework for protein quantitation in bottom-up MS based proteomics" (Karpievitch et al. Bioinformatics 2009). EigenMS normalisation is implemented as described in "Normalization of peak intensities in bottom-up MS-based proteomics using singular value decomposition." (Karpievitch et al. Bioinformatics 2009). |
| Authors: | Yuliya V Karpievitch, Tim Stuart and Sufyaan Mohamed |
| Maintainer: | Yuliya V Karpievitch <[email protected]> |
| License: | MIT |
| Version: | 1.23.0 |
| Built: | 2024-07-01 04:16:29 UTC |
| Source: | https://github.com/bioc/ProteoMM |
convert_log2 replaces 0's with NA's than does a log2 transformation Replacing 0's with NA's is the correct approach to Proteomics data analysis as 0's are not values that should be left in the data where no observation was made, see citation below. Karpievitch et al. 2009 "Normalization of peak intensities in bottom-up MS-based proteomics using singular value decomposition". PMID: 19602524 Karpievitch et al. 2009 "A statistical framework for protein quantitation in bottom-up MS-based proteomics". PMID: 19535538
convert_log2(mm, use_cols)convert_log2(mm, use_cols)
mm |
a dataframe of raw intensities in format: (# peptides)x(# samples+possibly peptide & protein information (metadata)) |
use_cols |
vector of column indecies that make up the intensities usually in sequential order but do not have to be user is responsible for making sure that specified columns are indeed numeric and correspond to intensities for each sample |
matrix of log2 transforemd intensities where 0's were replaced with NA's prior to transformation
data(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # 0's replaced with NAs and # log2 transnform applieddata(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # 0's replaced with NAs and # log2 transnform applied
First portion of EigenMS: Identify eigentrends attributable to bias, allow the user to adjust the number (with causion! if desired) before normalizing with eig_norm2. Ref: "Normalization of peak intensities in bottom-up MS-based proteomics using singular value decomposition" Karpievitch YV, Taverner T, et al. 2009, Bioinformatics Ref: "Metabolomics data normalization with EigenMS" Karpievitch YK, Nikolic SB, Wilson R, Sharman JE, Edwards LM 2014, PLoS ONE
eig_norm1(m, treatment, prot.info, write_to_file = "")eig_norm1(m, treatment, prot.info, write_to_file = "")
m |
number of peptides x number of samples matrix of log-transformed expression data, metadata not included in this matrix |
treatment |
either a single factor indicating the treatment group of each sample i.e. [1 1 1 1 2 2 2 2...] or a data frame of factors, eg: treatment= data.frame(cbind(data.frame(Group), data.frame(Time)) |
prot.info |
2+ colum data frame, pepID, prID columns IN THAT ORDER. IMPORTANT: pepIDs must be unique identifiers and will be used as Row Names If normalizing non-proteomics data, create a column such as: paste('ID_',seq_len(num_rows), sep=”) Same can be dome for ProtIDs, these are not used for normalization but are kept for future analyses |
write_to_file |
if a string is passed in, 'complete' peptides (peptides with NO missing observations) will be written to that file name |
A structure with multiple components
initial parameters passed into the function, returned for future reference
matrices produced by SVD
matrix of peptides that can be normalized, i.e. have enough observations for ANOVA
number of factors passed in
number of unique treatment facotr combinations, eg: Factor A: a a a a c c c c Factor B: 1 1 2 2 1 1 2 2 then: n.treatment = 2; n.u.treatment = 4
number of bias trends identified
names/IDs of peptides in variable 'pres'
complete peptides with no missing values, these were used to compute SVD
trends automatically identified in raw data, can be plotted at a later time
scores for each bias trend, eigenvalues
number of complete peptides with no missing observations
data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer name spaces # if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # 3 samples for CG and 3 for mCG grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(123) # Bias trends are determined via a permutaion, results may # vary slightly if a different seed is used, such as when set.seed() # function is not used mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1)data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer name spaces # if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # 3 samples for CG and 3 for mCG grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(123) # Bias trends are determined via a permutaion, results may # vary slightly if a different seed is used, such as when set.seed() # function is not used mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1)
Eliminate the effects of systematic bias identified in eig_norm1() Ref: "Normalization of peak intensities in bottom-up MS-based proteomics using singular value decomposition" Karpievitch YV, Taverner T et al. 2009, Bioinformatics Ref: "Metabolomics data normalization with EigenMS" Karpievitch YK, Nikolic SB, Wilson R, Sharman JE, Edwards LM Submitted to PLoS ONE.
eig_norm2(rv)eig_norm2(rv)
rv |
return value from the eig_norm1 if user wants to change the number of bias trends that will be eliminated h.c in rv should be updates to the desired number |
A structure with multiple components
matrix of normalized abundances with 2 columns of protein and peptdie names
matrix of normalized abundances, no extra columns
trends found in raw data, bias trends up to h.c
trends in normalized data, if one wanted to plot at later time
peptides excluded due to not enough peptides or exception in fitting a linear model
data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer name # spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(123) # set for repoducubility of eig_norm1 mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1)data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer name # spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(123) # set for repoducubility of eig_norm1 mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1)
Compute PI - proportion of observations missing completely at random
eigen_pi(m, toplot = TRUE)eigen_pi(m, toplot = TRUE)
m |
matrix of abundances, numsmaples x numpeptides |
toplot |
TRUE/FALSE plot mean vs protportion missing curve and PI |
pi estimate of the proportion of observations missing completely at random
Contributed by Shelley Herbrich & Tom Taverner for Karpievitch et al. 2009
data(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) my.pi = eigen_pi(m_logInts, toplot=TRUE)data(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) my.pi = eigen_pi(m_logInts, toplot=TRUE)
Log-likelihood test for independence & goodness of fit. g.test() performs Williams' and Yates' correction; Monte Carlo simulation of p-values, via gtestsim.c. MC requires recompilation of R. Written by Peter Hurd (V3.3 Pete Hurd Sept 29 2001, phurd AT ualberta.ca). Yuliya Karpievitch added comments for ease of understanding and incorporated into ProteoMM. G & q calculation from Sokal & Rohlf (1995) Biometry 3rd ed., TOI Yates correction taken from Mike Camanns 2x2 G-test function, GOF Yates correction as described in Zar (2000), more stuff taken from ctest's chisq.test().
g.test(x, y = NULL, correct = "none", p = rep(1/length(x), length(x)))g.test(x, y = NULL, correct = "none", p = rep(1/length(x), length(x)))
x |
vector of boolean values corresponding to presence & absence eg: c(TRUE, TRUE, FALSE, FALSE) for present present absent absent values. Order of TRUE/FALSE does not matter, can be used interchangeably. Same length as parameter y |
y |
vector treatments (factor) corresponding to values in x, same length as x eg: as.factor(c('grp1;, 'grp1', 'grp2', 'grp2')) |
correct |
correction to apply, options: "yates", "williams", "none" default: "none" NOTE: in ProteoMM we only tested & used correction = "none" |
p |
default: rep(1/length(x), length(x)), used in Yates correction NOTE: in ProteoMM we only tested & used the default parameter value |
htest object the following variables
value of the G statistic produced by g test
degrees of freedom of the test
p-value
method used to produce statistic and p-value
data passed in to the function
matrix of observed counts
matrix of expected counts
g.test(c(TRUE, TRUE, FALSE, FALSE), as.factor(c('grp1', 'grp1', 'grp2', 'grp2')))g.test(c(TRUE, TRUE, FALSE, FALSE), as.factor(c('grp1', 'grp1', 'grp2', 'grp2')))
Function get_presAbs_prots() produces a subset of protein meta data and intencities for multiple datasets pass in as a list. If a single dataset is passed in (list of length one) it will be processed in the same way as longer lists.
get_presAbs_prots(mm_list, prot.info, protnames_norm, prot_col_name)get_presAbs_prots(mm_list, prot.info, protnames_norm, prot_col_name)
mm_list |
list of matrices of intensities for each experiment. Dimentions: numpeptides x numsamples different for each dataset. |
prot.info |
list of protein and peptide metadata/mappings for each matrix in mm_list, data.frames "parallel" to matrices in mm_list. |
protnames_norm |
list of protein pdentifies to be used to determine peptides that will be placed into Presence/Absence analysis category due to too many missing peptides. Taken from the return value from eig_norm2(). |
prot_col_name |
column name (string) that will be used to get ProteinIDs in the raw data matrices |
list of lists of length 2
list of intecities in the same order and of the same length as the number of datasets that were passed into the function
list of protein metadata in the same order and of the same length as the number of datasets that as were passed into the function
# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2)# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2)
A dataset containing the protein and petide information and peptide-level intensities for 6 samples: 3 CG and 3 mCG groups. There are 69 proteins. The columns are as follows:
data(hs_peptides)data(hs_peptides)
A data frame with 695 rows and 13 colummns, compiring 7 columns of metadata and 6 columns of peptide intensities. 69 proteins.
Sequence - peptide sequence - randomly chosen from a larger list of sequences
MatchedID - numeric ID that links proteins in the two datasets, unnecessary if datasets are for the same species
ProtID - protein ID, artificial protein ID, eg. Prot1, Prot2, ...
GeneID - gene ID, artificial gene ID, eg. Gene1, Gene2, ...
ProtName - artificial Protein Name
ProtIDLong - long protein ID, full protein name, here artificially simulated
GeneIDLong - long gene ID, full gene name, here artificially simulated
CG1 - raw intensity column for sample 1 in CG group
CG2 - raw intensity column for sample 2 in CG group
CG3 - raw intensity column for sample 3 in CG group
mCG1 - raw intensity column for sample 1 in mCG group
mCG2 - raw intensity column for sample 2 in mCG group
mCG3 - raw intensity column for sample 3 in mCG group
Subdivide a data frame of protein intensities and metadata into intensities only. No row names will be provided.
make_intencities(mm, use_cols)make_intencities(mm, use_cols)
mm |
data frame of metadata and intensities as a single data frame |
use_cols |
column numbers to subset and return, no range checking no range checking on the column indeces is performed |
m_ints data frame of intensities only
data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses outer name # spaces if variable is undefined m_logInts = make_intencities(mm_peptides, intsCols)data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses outer name # spaces if variable is undefined m_logInts = make_intencities(mm_peptides, intsCols)
Subdivide a data frame of protein metadata and intensities into a data frame of meta data only
make_meta(mm, use_cols)make_meta(mm, use_cols)
mm |
data frame of metadata and intensities as a signle data frame |
use_cols |
column numbers to subset and return, no range checking on the column indeces is performed |
m_ints data frame of intensities only
data(mm_peptides) head(mm_peptides) metaCols = 1:7 # reusing this variable m_prot.info = make_meta(mm_peptides, metaCols)data(mm_peptides) head(mm_peptides) metaCols = 1:7 # reusing this variable m_prot.info = make_meta(mm_peptides, metaCols)
Makes a string linear model formula suitable for the right hand side of the equasion passed into lm()
makeLMFormula(eff, var_name = "")makeLMFormula(eff, var_name = "")
eff |
treatment group ordering for all samples being anlysed. Single factor with 2+ teatment groups. Used to generate formula and contrasts for lm(). |
var_name |
string variable name to use in the formula |
eig_norm1 and eig_norm2 Here we incorporate the model matrix from EigenMS normalization to find the significant trends in the matrix of residuals.
data structure with linea model formula and contrasts
Lienar model formula suitable for right hand side of ' ~' in lm(), ~ is not included int eh formula
contrasts for lm(), here sum-to-zero constraint only
grps = as.factor(c('CG', 'CG', 'CG', 'mCG', 'mCG', 'mCG')) makeLMFormula(grps, 'TREATS')grps = as.factor(c('CG', 'CG', 'CG', 'mCG', 'mCG', 'mCG')) makeLMFormula(grps, 'TREATS')
Impute missing values based on information from multiple peptides within a protein Expects the data to be filtered to contain at least one observation per treatment group. For experiments with lower overall abundaneces such as multiplexed experiments check if the imputed value is below 0, if so value is reimputed untill it is above 0.
MBimpute(mm, treatment, prot.info, pr_ppos = 2, my.pi = 0.05, compute_pi = FALSE)MBimpute(mm, treatment, prot.info, pr_ppos = 2, my.pi = 0.05, compute_pi = FALSE)
mm |
number of peptides x number of samples matrix of intensities |
treatment |
vector indicating the treatment group of each sample eg as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) or c(1,1,1,1,2,2,2,2) |
prot.info |
protein metadata, 2+ columns: peptide IDs, protein IDs, etc |
pr_ppos |
column index for protein ID in prot.info |
my.pi |
PI value, estimate of the proportion of peptides missign completely at random, as compared to censored at lower abundance levels default values of 0.05 is usually reasoanble for missing completely at random values in proteomics data |
compute_pi |
TRUE/FALSE (default=FALSE) estimate Pi is set to TRUE, otherwise use the provided value. We consider Pi=0.05 a reasonable estimate for onservations missing completely at random in proteomics experiments. Thus values is set to NOT estimate Pi by default. Note: spline smoothing can sometimes produce values of Pi outside the range of possible values. |
A structure with multiple components
number of peptides x m matrix of peptides with no missing data
imputed protein info, 2+ columns: peptide ID, protein IDs, etc Dimentions should be the same as passed in
data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses outer name spaces # if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(125) # if nto set every time results will be different imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE)data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses outer name spaces # if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(125) # if nto set every time results will be different imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE)
A dataset containing the protein and petide information and peptide-level intensities for 6 samples: 3 CG and 3 mCG groups. There are 69 proteins. The columns are as follows:
data(mm_peptides)data(mm_peptides)
A data frame with 1102 rows and 13 colummns, compiring 7 columns of metadata and 6 columns of peptide intensities. 69 proteins.
Sequence - peptide sequence - randomly chosen from a larger list of sequences
MatchedID - numeric ID that links proteins in the two datasets, unnecessary if datasets are for the same species
ProtID - protein ID, artificial protein ID, eg. Prot1, Prot2, ...
GeneID - gene ID, artificial gene ID, eg. Gene1, Gene2, ...
ProtName - artificial Protein Name
ProtIDLong - long protein ID, full protein name, here artificially simulated
GeneIDLong - long gene ID, full gene name, here artificially simulated
CG1 - raw intensity column for sample 1 in CG group
CG2 - raw intensity column for sample 2 in CG group
CG3 - raw intensity column for sample 3 in CG group
mCG1 - raw intensity column for sample 1 in mCG group
mCG2 - raw intensity column for sample 2 in mCG group
mCG3 - raw intensity column for sample 3 in mCG group
Model-Based differential expression analysis is performed on peptide level as desribed in Karpievitch et al. 2009 "A statistical framework for protein quantitation in bottom-up MS-based proteomics" Bioinformatics.
peptideLevel_DE(mm, treatment, prot.info, pr_ppos = 2)peptideLevel_DE(mm, treatment, prot.info, pr_ppos = 2)
mm |
m x n matrix of intensities, num peptides x num samples |
treatment |
vector indicating the treatment group of each sample ie [1 1 1 1 2 2 2 2...] |
prot.info |
2+ colum data frame of peptide ID, protein ID, etc. columns |
pr_ppos |
- column index for protein ID in prot.info. Can restrict to be #2... |
A data frame with the following columns:
protein identification information taken from prot.info, 1 column used to identify proteins
fold change
p-value for the comparison between 2 groups (2 groups only here)
Benjamini-Hochberg adjusted p-values
data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer # name spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) # results rarely vary due to the random seed for EigenMS mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(131) # important to reproduce the results later imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, mm_m_ints_norm$normalized[,metaCols], pr_ppos=2)data(mm_peptides) head(mm_peptides) # different from parameter names as R uses outer # name spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) # results rarely vary due to the random seed for EigenMS mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(131) # important to reproduce the results later imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, mm_m_ints_norm$normalized[,metaCols], pr_ppos=2)
Presence/Absence peptide-level analysis uses all peptides for a protein as IID to produce 1 p-value across multiple (2+) datasets. Significance is estimated using a g-test which is suitable for two treatment groups only.
peptideLevel_PresAbsDE(mm, treatment, prot.info, pr_ppos = 2)peptideLevel_PresAbsDE(mm, treatment, prot.info, pr_ppos = 2)
mm |
m x n matrix of intensities, num peptides x num samples |
treatment |
vector indicating the treatment group of each sample ie [1 1 1 1 2 2 2 2...] |
prot.info |
2+ colum data frame of peptide ID, protein ID, etc columns |
pr_ppos |
- column index for protein ID in prot.info. Can restrict to be #2... |
A list of length two items:
protein identification information taken from prot.info, a column used to identify proteins
Approximation of the fold change computed as percent missing observations group 1 munis in percent missing observations group 2
p-value for the comparison between 2 groups (2 groups only here)
Benjamini-Hochberg adjusted p-values
statistic returned by the g-test, not very useful as depends on the direction of the test and can produce all 0's
number of peptides within a protein
all columns of metadata from the passed in matrix
# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 # different from parameter names as R # uses outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(137) # different seed for different organism hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2) presAbs_de = peptideLevel_PresAbsDE(presAbs_dd[[1]][[1]], grps, presAbs_dd[[2]][[1]], pr_ppos=2)# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 # different from parameter names as R # uses outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(137) # different seed for different organism hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2) presAbs_de = peptideLevel_PresAbsDE(presAbs_dd[[1]][[1]], grps, presAbs_dd[[2]][[1]], pr_ppos=2)
Plot peptide trends for a protein
plot_1prot(mm, prot.info, prot_to_plot, prot_to_plot_col, gene_name, gene_name_col, colors, mylabs)plot_1prot(mm, prot.info, prot_to_plot, prot_to_plot_col, gene_name, gene_name_col, colors, mylabs)
mm |
matrix of raw intensities |
prot.info |
metadata for the intensities in mm |
prot_to_plot |
protein ID to plot |
prot_to_plot_col |
protein ID column index |
gene_name |
gene ID to plot |
gene_name_col |
gene ID to plot column index |
colors |
what colors to plot peptide abundances as, most commonly should be treatment groups |
mylabs |
sample labels to be plotted on x-axis |
Nil
Plot Raw, Normalized and Normalized & Imputed peptide trends for a protein
plot_3_pep_trends_NOfile(mm, prot.info, sorted_norm_m, sorted_prot.info, imp_mm, imp_prot.info, prot_to_plot, prot_to_plot_col, gene_name, gene_name_col, mylabs)plot_3_pep_trends_NOfile(mm, prot.info, sorted_norm_m, sorted_prot.info, imp_mm, imp_prot.info, prot_to_plot, prot_to_plot_col, gene_name, gene_name_col, mylabs)
mm |
matrix of raw intensities |
prot.info |
metadata for the intensities in mm |
sorted_norm_m |
normalized intensities, possibly fewer than in mm due to filtering out peptides with fewer than one observation per treatment group |
sorted_prot.info |
metadata for the intensities in sorted_norm_m |
imp_mm |
imputed intensities (post normalization) |
imp_prot.info |
metadata for the imputed intensities in imp_mm |
prot_to_plot |
protein ID to plot |
prot_to_plot_col |
protein ID column index |
gene_name |
gene ID to plot |
gene_name_col |
gene ID to plot column index |
mylabs |
sample labels to be plotted on x-axis |
Nil
data("hs_peptides") # loads variable hs_peptides intsCols = 8:13 # column indeces that contain intensities m_logInts = make_intencities(hs_peptides, intsCols) # replace 0's with NA's as NA's are more appropriate # for anlysis and log2 transform m_logInts = convert_log2(m_logInts) # column indices that contain metadata such as protein IDs and sequences metaCols = 1:7 m_prot.info = make_meta(hs_peptides, metaCols) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c = 2 # looks like there are 2 bias trends at least hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,metaCols] hs_norm_m = hs_m_ints_norm$normalized[,intsCols] set.seed(125) imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=3, my.pi=0.05, compute_pi=FALSE) mylabs = c( 'CG','CG','CG', 'mCG','mCG','mCG') prot_to_plot = 'Prot32' # 43 gene_to_plot = 'Gene32' plot_3_pep_trends_NOfile(as.matrix(hs_m_ints_eig1$m), hs_m_ints_eig1$prot.info, as.matrix(hs_norm_m), hs_prot.info, imp_hs$y_imputed, imp_hs$imp_prot.info, prot_to_plot, 3, gene_to_plot, 4, mylabs)data("hs_peptides") # loads variable hs_peptides intsCols = 8:13 # column indeces that contain intensities m_logInts = make_intencities(hs_peptides, intsCols) # replace 0's with NA's as NA's are more appropriate # for anlysis and log2 transform m_logInts = convert_log2(m_logInts) # column indices that contain metadata such as protein IDs and sequences metaCols = 1:7 m_prot.info = make_meta(hs_peptides, metaCols) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c = 2 # looks like there are 2 bias trends at least hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,metaCols] hs_norm_m = hs_m_ints_norm$normalized[,intsCols] set.seed(125) imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=3, my.pi=0.05, compute_pi=FALSE) mylabs = c( 'CG','CG','CG', 'mCG','mCG','mCG') prot_to_plot = 'Prot32' # 43 gene_to_plot = 'Gene32' plot_3_pep_trends_NOfile(as.matrix(hs_m_ints_eig1$m), hs_m_ints_eig1$prot.info, as.matrix(hs_norm_m), hs_prot.info, imp_hs$y_imputed, imp_hs$imp_prot.info, prot_to_plot, 3, gene_to_plot, 4, mylabs)
Function plots fold changes and p-values as a volcano plot. Two lines are plotted for the p-value cutoff at p = PV_cutoff (solid line) and p = 0.1 (dashed line).
plot_volcano(FC, PV, FC_cutoff = 2, PV_cutoff = 0.05, figtitle = "")plot_volcano(FC, PV, FC_cutoff = 2, PV_cutoff = 0.05, figtitle = "")
FC |
vector of fold changes |
PV |
vctor of p-values, same lenght as FC |
FC_cutoff |
fold change cutoff where to draw vertical cutoff lines, default = 2 |
PV_cutoff |
p-value cutoff where to draw a horisontal cutoff line, default ==.05 |
figtitle |
title to display at the top of the figure, default = ” |
Nil
data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as # R uses outer name spaces if variable is undefined metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # Normalize data grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(123) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected # Impute missing values mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) # needed for reproducibility of imputation imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, imp_mm$imp_prot.info, pr_ppos=2) plot_volcano(DE_res$FC, DE_res$BH_P_val, FC_cutoff=1.5, PV_cutoff=.05, figtitle='Mouse DE')data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as # R uses outer name spaces if variable is undefined metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # Normalize data grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(123) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected # Impute missing values mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) # needed for reproducibility of imputation imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, imp_mm$imp_prot.info, pr_ppos=2) plot_volcano(DE_res$FC, DE_res$BH_P_val, FC_cutoff=1.5, PV_cutoff=.05, figtitle='Mouse DE')
Function plots fold changes and p-values as a volcano plot. Two lines are plotted for the p-value cutoff at p = PV_cutoff (solid line) and p = 0.1 (dashed line).
plot_volcano_wLab(FC, PV, ProtID, FC_cutoff = 2, PV_cutoff = 0.05, figtitle = "")plot_volcano_wLab(FC, PV, ProtID, FC_cutoff = 2, PV_cutoff = 0.05, figtitle = "")
FC |
vector of fold changes |
PV |
vector of p-values, same lenght as FC |
ProtID |
vector of protein IDs, can be gene IDs, same lenght as FC & PV. Namaes in this vector will be displayed in the volcano plot for differentially expressed proteins for this reason short names are preferred. |
FC_cutoff |
fold change cutoff where to draw vertical cutoff lines, default = 2 |
PV_cutoff |
p-value cutoff where to draw a horisontal cutoff line, default ==.05 |
figtitle |
title to display at the top of the figure, default = ” |
Nil
data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as # R uses outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # Normalize data grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected # Impute missing values mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, imp_mm$imp_prot.info, pr_ppos=2) plot_volcano_wLab(DE_res$FC, DE_res$BH_P_val, DE_res$ProtID, FC_cutoff=1.5, PV_cutoff=.05, figtitle='Mouse DE')data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as # R uses outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) # Normalize data grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected # Impute missing values mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) DE_res = peptideLevel_DE(imp_mm$y_imputed, grps, imp_mm$imp_prot.info, pr_ppos=2) plot_volcano_wLab(DE_res$FC, DE_res$BH_P_val, DE_res$ProtID, FC_cutoff=1.5, PV_cutoff=.05, figtitle='Mouse DE')
Multi-Matrix Differential Expression Analysis computes Model-Based statistics for each dataset, the sum of individual statistics is the final statistic. The significance is determined via a permutation test which computed the same statistics and sums them after permuting the values across treatment groups. As is outlined in Karpievitch et al. 2018. Important to set the random number generator seed for reprodusibility with set.seed() function.
prot_level_multi_part(mm_list, treat, prot.info, prot_col_name, nperm = 500, dataset_suffix)prot_level_multi_part(mm_list, treat, prot.info, prot_col_name, nperm = 500, dataset_suffix)
mm_list |
list of matrices for each experiment, length = number of datasets to compare internal dataset dimentions: numpeptides x numsamples for each dataset |
treat |
list of data frames with treatment information to compute the statistic in same order as mm_list |
prot.info |
list of protein and peptide mapping for each matrix in mm_list, in same order as mm_list |
prot_col_name |
column name in prot.info that contains protein identifiers that link all datasets together. Not that Protein IDs will differ across different organizms and cannot be used as the linking identifier. Function match_linker_ids() produces numeric identifyers that link all datasets together |
nperm |
number of permutations, default = 500, this will take a while, test code with fewer permutations |
dataset_suffix |
vector of character strings that corresponds to the dataset being analysed. Same length as mm_list. Names will be appended to the columns names that will be generated for each analysed dataset. For example, if analysing mouse and human data this vector may be: c('Mouse', 'Human') |
data frame with the following columns
Column containing the protien IDs used to link proteins across datasets
Average fold change across all datasets
Permutation-based p-valu for the differences between the groups
Multiple testing adjusted p-values
Statistic computed as a a sum of statistics produced for each dataset
all columns passed into the function for the 1st dataset in the list
Fold changes for individual datasets, these values should average to the FC above. As many columns as there are datasets being analyzed.
p-values for individual datasets. As many columns as there are datasets being analyzed.
Multiple testing adjusted p-values for individual datasets. As many columns as there are datasets being analyzed.
Number of peptides presents in each protien for each dataset. As many columns as there are datasets being analyzed.
# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps, prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) # Needed for reprodicibility of results imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(1237) # needed for reproducibility hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,1:7] hs_norm_m = hs_m_ints_norm$normalized[,8:13] set.seed(125) # or any value, ex: 12345 imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Multi-Matrix Model-based differential expression analysis # Set up needed variables mms = list() treats = list() protinfos = list() mms[[1]] = imp_mm$y_imputed mms[[2]] = imp_hs$y_imputed treats[[1]] = grps treats[[2]] = grps protinfos[[1]] = imp_mm$imp_prot.info protinfos[[2]] = imp_hs$imp_prot.info nperm = 50 # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(131) # needed for reproducibility comb_MBDE = prot_level_multi_part(mm_list=mms, treat=treats, prot.info=protinfos, prot_col_name='ProtID', nperm=nperm, dataset_suffix=c('MM', 'HS')) # Analysis for proteins only present in mouse, # there are no proteins suitable for # Model-Based analysis in human dataset subset_data = subset_proteins(mm_list=mms, prot.info=protinfos, 'MatchedID') mm_dd_only = subset_data$sub_unique_mm_list[[1]] hs_dd_only = subset_data$sub_unique_mm_list[[2]] protinfos_mm_dd = subset_data$sub_unique_prot.info[[1]] DE_mCG_CG_mm_dd = peptideLevel_DE(mm_dd_only, grps, prot.info=protinfos_mm_dd, pr_ppos=2)# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps, prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(125) # Needed for reprodicibility of results imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 # different from parameter names as R uses # outer name spaces if variable is undefined metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(1237) # needed for reproducibility hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,1:7] hs_norm_m = hs_m_ints_norm$normalized[,8:13] set.seed(125) # or any value, ex: 12345 imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Multi-Matrix Model-based differential expression analysis # Set up needed variables mms = list() treats = list() protinfos = list() mms[[1]] = imp_mm$y_imputed mms[[2]] = imp_hs$y_imputed treats[[1]] = grps treats[[2]] = grps protinfos[[1]] = imp_mm$imp_prot.info protinfos[[2]] = imp_hs$imp_prot.info nperm = 50 # ATTENTION: SET RANDOM NUMBER GENERATOR SEED FOR REPRODUCIBILITY !! set.seed(131) # needed for reproducibility comb_MBDE = prot_level_multi_part(mm_list=mms, treat=treats, prot.info=protinfos, prot_col_name='ProtID', nperm=nperm, dataset_suffix=c('MM', 'HS')) # Analysis for proteins only present in mouse, # there are no proteins suitable for # Model-Based analysis in human dataset subset_data = subset_proteins(mm_list=mms, prot.info=protinfos, 'MatchedID') mm_dd_only = subset_data$sub_unique_mm_list[[1]] hs_dd_only = subset_data$sub_unique_mm_list[[2]] protinfos_mm_dd = subset_data$sub_unique_prot.info[[1]] DE_mCG_CG_mm_dd = peptideLevel_DE(mm_dd_only, grps, prot.info=protinfos_mm_dd, pr_ppos=2)
Multi-Matrix Presence Absence Analysis computes Model-Based statistics for each dataset and sums them up to produce the final statistic. The significance is determined via a permutation test which computes the same statistics and sums them after permuting the values across treatment groups, as is outlined in Karpievitch et al. 2018. Whenever possible proteins should be analysed using the Model-Based Differential Expression Analysis due to higher statistical power over the Presence Absence analysis.
prot_level_multiMat_PresAbs(mm_list, treat, prot.info, prot_col_name, nperm = 500, dataset_suffix)prot_level_multiMat_PresAbs(mm_list, treat, prot.info, prot_col_name, nperm = 500, dataset_suffix)
mm_list |
list of matrices of intensities for each experiment, dimentions: numpeptides x numsamples |
treat |
list of data frames with treatment information to compute the statistic, parallel to mm_list and prot.info |
prot.info |
list of protein metadata for each matrix in mm_list, data.frame parallel to mm_list and treat |
prot_col_name |
column names present in all datasets that identifies protein IDs across all datasets |
nperm |
number of permutations |
dataset_suffix |
a list of strings that will be appended to the column names for FC, PV, BHPV and numebers of peptides |
a data frame with the following columns:
protein metadata, peptide sequence if was passed in as one of the columns is the first peptide equence encountered in the data for that protein
Avegares across all datasets of the approximation of the fold change computed as percent missing observations group 1 munis in percent missing observations group 2 in peptideLevel_PresAbsDE() function
p-value for the comparison between 2 groups (2 groups only here) obtained from a permutation test
Benjamini-Hochberg adjusted p-values
statistic returned by the g-test and summed across all datasets, not very useful as depends on the direction of the test and can produce all 0's
column containing ptoein identifiers across all datasets
Approximation of the fold change computed as percent missing observations group 1 munis in percent missing observations group 2 in peptideLevel_PresAbsDE() function
p-values produced by g-test for individual datasets
adjusted p-values produced by g-test for individual datasets
number of peptides observed for each protein in each of the datasets
# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(hs_peptides, intsCols) m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(137) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2) ints_presAbs = list() protmeta_presAbs = list() ints_presAbs[[1]] = presAbs_dd[[1]][[1]] # Mouse ints_presAbs[[2]] = presAbs_dd[[1]][[2]] # HS protmeta_presAbs[[1]] = presAbs_dd[[2]][[1]] protmeta_presAbs[[2]] = presAbs_dd[[2]][[2]] treats = list() treats[[1]] = grps treats[[2]] = grps subset_presAbs = subset_proteins(mm_list=ints_presAbs, prot.info=protmeta_presAbs, 'MatchedID') nperm = 50 # set to 500+ for publication set.seed(275937) presAbs_comb = prot_level_multiMat_PresAbs( mm_list=subset_presAbs$sub_mm_list, treat=treats, prot.info=subset_presAbs$sub_prot.info, prot_col_name='MatchedID', nperm=nperm, dataset_suffix=c('MM', 'HS') ) plot_volcano(presAbs_comb$FC, presAbs_comb$BH_P_val, FC_cutoff=.5, PV_cutoff=.05, 'Combined Pres/Abs CG vs mCG')# Load mouse dataset data(mm_peptides) head(mm_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(135) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 m_logInts = make_intencities(hs_peptides, intsCols) m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(137) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) # Set up for presence/absence analysis raw_list = list() norm_imp_prot.info_list = list() raw_list[[1]] = mm_m_ints_eig1$m raw_list[[2]] = hs_m_ints_eig1$m norm_imp_prot.info_list[[1]] = mm_m_ints_eig1$prot.info norm_imp_prot.info_list[[2]] = hs_m_ints_eig1$prot.info protnames_norm_list = list() protnames_norm_list[[1]] = unique(mm_m_ints_norm$normalized$MatchedID) protnames_norm_list[[2]] = unique(hs_m_ints_norm$normalized$MatchedID) presAbs_dd = get_presAbs_prots(mm_list=raw_list, prot.info=norm_imp_prot.info_list, protnames_norm=protnames_norm_list, prot_col_name=2) ints_presAbs = list() protmeta_presAbs = list() ints_presAbs[[1]] = presAbs_dd[[1]][[1]] # Mouse ints_presAbs[[2]] = presAbs_dd[[1]][[2]] # HS protmeta_presAbs[[1]] = presAbs_dd[[2]][[1]] protmeta_presAbs[[2]] = presAbs_dd[[2]][[2]] treats = list() treats[[1]] = grps treats[[2]] = grps subset_presAbs = subset_proteins(mm_list=ints_presAbs, prot.info=protmeta_presAbs, 'MatchedID') nperm = 50 # set to 500+ for publication set.seed(275937) presAbs_comb = prot_level_multiMat_PresAbs( mm_list=subset_presAbs$sub_mm_list, treat=treats, prot.info=subset_presAbs$sub_prot.info, prot_col_name='MatchedID', nperm=nperm, dataset_suffix=c('MM', 'HS') ) plot_volcano(presAbs_comb$FC, presAbs_comb$BH_P_val, FC_cutoff=.5, PV_cutoff=.05, 'Combined Pres/Abs CG vs mCG')
Subset proteins into ones common to all datasets passed into the function and unique to each dataset. Note: for 3+ datasets no intermediate combinations of proteins are returned, only proteins common to all datasets, the rest are returned as unique to each dataset.
subset_proteins(mm_list, prot.info, prot_col_name)subset_proteins(mm_list, prot.info, prot_col_name)
mm_list |
list of matrices for each experiment, length = number of datasets to compare internal dataset dimentions: numpeptides x numsamples for each dataset |
prot.info |
list of protein and peptide mapping for each matrix in mm_list, in same order as mm_list |
prot_col_name |
column name in prot.info that contains protein identifiers that link all datasets together. Not that Protein IDs will differ across different organizms and cannot be used as the linking identifier. Function match_linker_ids() produces numeric identifyers that link all datasets together |
data frame with the following columns
list of dataframes of intensities for each of the datasets passed in with proteins present in all datasets
list of dataframes of metadata for each of the datasets passed in with proteins present in all datasets. Same order as sub_mm_list
list of dataframes of intensities not found in all datasets
ist of dataframes of metadata not found in all datasets
list of protein IDs commnon to all datasets
# Load mouse dataset data(mm_peptides) head(mm_peptides) # different from parameter names as R uses # outer name spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(173) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(131) imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,1:7] hs_norm_m = hs_m_ints_norm$normalized[,8:13] set.seed(131) imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Multi-Matrix Model-based differential expression analysis # Set up needed variables mms = list() treats = list() protinfos = list() mms[[1]] = imp_mm$y_imputed mms[[2]] = imp_hs$y_imputed treats[[1]] = grps treats[[2]] = grps protinfos[[1]] = imp_mm$imp_prot.info protinfos[[2]] = imp_hs$imp_prot.info subset_data = subset_proteins(mm_list=mms, prot.info=protinfos, 'MatchedID') mms_mm_dd = subset_data$sub_unique_mm_list[[1]] protinfos_mm_dd = subset_data$sub_unique_prot.info[[1]] # DIfferential expression analysis for mouse specific protiens DE_mCG_CG_mm_dd = peptideLevel_DE(mms_mm_dd, grps, prot.info=protinfos_mm_dd, pr_ppos=2)# Load mouse dataset data(mm_peptides) head(mm_peptides) # different from parameter names as R uses # outer name spaces if variable is undefined intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(mm_peptides, intsCols) # will reuse the name m_prot.info = make_meta(mm_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) set.seed(173) mm_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) mm_m_ints_eig1$h.c # check the number of bias trends detected mm_m_ints_norm = eig_norm2(rv=mm_m_ints_eig1) mm_prot.info = mm_m_ints_norm$normalized[,1:7] mm_norm_m = mm_m_ints_norm$normalized[,8:13] set.seed(131) imp_mm = MBimpute(mm_norm_m, grps, prot.info=mm_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Load human dataset data(hs_peptides) head(hs_peptides) intsCols = 8:13 metaCols = 1:7 # reusing this variable m_logInts = make_intencities(hs_peptides, intsCols) # will reuse the name m_prot.info = make_meta(hs_peptides, metaCols) m_logInts = convert_log2(m_logInts) grps = as.factor(c('CG','CG','CG', 'mCG','mCG','mCG')) hs_m_ints_eig1 = eig_norm1(m=m_logInts,treatment=grps,prot.info=m_prot.info) hs_m_ints_eig1$h.c # check the number of bias trends detected hs_m_ints_norm = eig_norm2(rv=hs_m_ints_eig1) hs_prot.info = hs_m_ints_norm$normalized[,1:7] hs_norm_m = hs_m_ints_norm$normalized[,8:13] set.seed(131) imp_hs = MBimpute(hs_norm_m, grps, prot.info=hs_prot.info, pr_ppos=2, my.pi=0.05, compute_pi=FALSE) # Multi-Matrix Model-based differential expression analysis # Set up needed variables mms = list() treats = list() protinfos = list() mms[[1]] = imp_mm$y_imputed mms[[2]] = imp_hs$y_imputed treats[[1]] = grps treats[[2]] = grps protinfos[[1]] = imp_mm$imp_prot.info protinfos[[2]] = imp_hs$imp_prot.info subset_data = subset_proteins(mm_list=mms, prot.info=protinfos, 'MatchedID') mms_mm_dd = subset_data$sub_unique_mm_list[[1]] protinfos_mm_dd = subset_data$sub_unique_prot.info[[1]] # DIfferential expression analysis for mouse specific protiens DE_mCG_CG_mm_dd = peptideLevel_DE(mms_mm_dd, grps, prot.info=protinfos_mm_dd, pr_ppos=2)
Surrogate Variable Analysis function used internatlly by eig_norm1 and eig_norm2 Here we incorporate the model matrix from EigenMS normalization to find the significant trends in the matrix of residuals.
sva.id(dat, n.u.treatment, lm.fm, B = 500, sv.sig = 0.05)sva.id(dat, n.u.treatment, lm.fm, B = 500, sv.sig = 0.05)
dat |
number of peptides/genes x number of samples matrix of expression data with no missing values |
n.u.treatment |
number of treatment groups |
lm.fm |
formular for treatment to be use on the right side of the call to stats::lm() as generated by makeLMFormula() |
B |
The number of null iterations to perform |
sv.sig |
The significance cutoff for the surrogate variables |
A data structure with the following values:
Number of significant surrogate variables
Significance for the returned surrogate variables