VIPER relies on the following R packages: Rcpp, RcppArmadillo, quadprog, glmnet. All packagess are hosted on CRAN.
install.packages("Rcpp")
install.packages("RcppArmadillo")
install.packages("quadprog")
install.packages("glmnet")VIPER can be installed from github directly as follows:
install.packages("devtools")
library(devtools)
install_github("ChenMengjie/VIPER")VIPER() is the main funtion that implements the imputation methods. Details about the steps can be found in later sections. To perform imputation, simply run:
library(VIPER)
data(grun)
res <- VIPER(gene.expression, num = 5000, percentage.cutoff = 0.1, minbool = FALSE, alpha = 1,
report = FALSE, outdir = NULL, prefix = NULL)Our method is not restricted to the units of measurement and is applicable to all normalized measurements such as RPM (reads per million reads), TPM (transcripts per kilobase per millions reads) or RPKM (reads per kilobase per millions reads).
Paramter num is the number of random sampled genes used to fit the penalized regression model to identify the set of candidate cells. The default value is 5000. To reduce the influence of missing values in the weight estimation, the nonnegative regression model is fitted using genes with a zero rate less than a certain threshold. The threshold is set using percentage.cutoff.
VIPER calls cv.glmnet() in glmnet to perform fitting cross validation. Two penalty levels are available for selection of penalty level lambda: lambda.min, the value of lambda that gives minimum mean cross-validated error, and lambda.1se, the value of lambda that gives the most regularized model such that error is within one standard error of the minimum. The default is lambda.1se, i.e., minbool = FALSE.
Paramter alpha sets the elastic net mixing parameter. The default value is 1, which is equivalent to a lasso model. We used alpha = 0.5 for elastic net.
Paramter report controls whether to save imputed data matrix in CSV files. The default value is FALSE. When report is TRUE, paramter outdir provide the directory to save the result files. Paramter prefix sets the prefix of the result files.
VIPER() returns the following list of values:
imputed_log: A p by n matrix of log transformed gene expression levels after imputation.
imputed: A p by n matrix of gene expression counts converted from imputed log transformed values.
sample_weights: A n by n matrix of estimated imputation weights. Each row represents a cell.
outliers: The indexes of cells that have no selected candidate neighbors according to the penalized regression model. The zero values in these cells are not imputed.
A tutorial with examples illustrating the usage of VIPER is available at:
https://github.com/ChenMengjie/VIPER/blob/master/VIPERUserguide.pdf
We have primarily focused on illustrating our method on selecting neighborhood cells for imputation. However, we acknowledge that all data examined in the manuscript contain a smaller number of cells than the number of genes. The accuracy of gene based imputation is likely dependent on the number of cells and will likely improve with increasing cell number. In contrast, the accuracy of cell based imputation is likely dependent on the number of genes. Subsequently, using genes to perform imputation may have added benefits for larger scRNAseq data where the number of cells exceeds the number of genes. We tested VIPER on several low coverage 10X and Drop-seq data, and we found gene-to-gene imputation could be better than cell-to-cell. To test this on a new dataset, you can perform the 2.1 analysis shown here: https://github.com/ChenMengjie/VIPER/blob/master/VIPERUserguide.pdf (with GeneNum=200, ZeroRate=0.7) to check which way is better. If gene-to-gene predictions obtain better R squares, we recommend to transpose the gene expression as input shown as follows:
library(VIPER)
res <- VIPER(t(gene.expression), num = 1000, percentage.cutoff = 0.5, minbool = FALSE, alpha = 0.5,
report = FALSE, outdir = NULL, prefix = NULL)Mengjie Chen (UChicago) [email protected]