---
title: "BioMM: Biological-informed Multi-stage Machine learning framework for phenotype prediction using omics data"
author:
 - name: Junfang Chen and Emanuel Schwarz
   affiliation: Central Institute of Mental Health, Heidelberg University, Germany  
date: "Modified: 17 July 2019. Compiled: `r format(Sys.Date(), '%d %b %Y')`"
output: 
  BiocStyle::html_document: 
    toc_float: false
    number_sections: true 
    href: BioMMtutorial.html 

vignette: >
   %\VignetteIndexEntry{BioMMtutorial}
   %\VignetteEngine{knitr::rmarkdown}
   %\VignetteEncoding{UTF-8}
---

```{r style, echo = FALSE, results = 'asis'}
BiocStyle::markdown()
```
```{r global_options, include=FALSE}  
knitr::opts_chunk$set(message=FALSE, error=FALSE, warning=FALSE, fig.width=8, 
fig.height=8)
options(width=100) 
```


# Overview
## Motivation
The identification of reproducible biological patterns from high-dimensional 
omics data is a key factor in understanding the biology of complex disease or 
traits. Incorporating prior biological knowledge into machine learning is an 
important step in advancing such research.

## Deliverables
We have implemented a biologically informed multi-stage machine learing 
framework termed __BioMM__ [1] specifically for phenotype prediction using 
omics-scale data based on biological prior information, for example, gene ontological pathways.   

**Features of BioMM in a nutshell**:   

1. Applicability for various omics data modalities.      
2. Prioritizing outcome-associated functional patterns.   
3. End-to-end prediction at the individual level based on biological 
pathway stratification.  
4. Possibility for extension to machine learning models of interest.   
5. Parallel computing. 

# Getting started  

## Installation  
*  Development version from Github (R 3.5):

```{r eval=FALSE}
install.packages("devtools")
library("devtools")
install_github("transbioZI/BioMM")
```

*  Install BioMM from Bioconductor (R 3.6):
```{r eval=FALSE}
if (!requireNamespace("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
# The following initializes usage of Bioc devel
BiocManager::install(version='devel')
BiocManager::install("BioMM")
```

Note that, two different parallel computing strategies are implemented: For BioMM package available on Github, the parallel R package is implemented. The BiocParallel R package is employed in Bioconductor BioMM package which allows the use of parallel computation across any system.

* Load required libraries
```{r loadPkg, eval=TRUE, results="hide"}
library(BioMM)
library(BiocParallel)
library(parallel)
library(ranger)
library(rms)
library(glmnet)
library(e1071)
library(pROC)
library(vioplot)
library(variancePartition)
library(CMplot)
```

# Omics data 
A wide range of genome-wide omics data is supported for the use of BioMM 
including whole-genome DNA methylation, gene expression and genome-wide SNP 
data. Other types of omics data that can map into pathways are also encouraging.  
For better understanding of the framework, we used a preprocessed genome-wide 
DNA methylation data with 26486 CpGs and 40 samples consisting of 20 controls 
and 20 patients. (0: healthy control and 1: patient) for demonstration.  

```{r studyData, eval=TRUE}
## Get DNA methylation data 
studyData <- readRDS(system.file("extdata", "/methylData.rds", 
                     package="BioMM"))
head(studyData[,1:5])
dim(studyData)
``` 

# Feature stratification
Features like CpGs, genes or SNPs can be mapped into pathways based on genomic location and pathway annotation, as implemented in the function `omics2pathlist()`. The examples of pathway databases are gene ontology (GO), Reactome and KEGG, which are widely used public pathway repositories. Gene ontological pathway is used in this tutorial.

```{r annotationFile, eval=TRUE}
## Load annotation data
featureAnno <- readRDS(system.file("extdata", "cpgAnno.rds", package="BioMM")) 
pathlistDB <- readRDS(system.file("extdata", "goDB.rds", package="BioMM")) 
head(featureAnno)
str(pathlistDB[1:3])
``` 

```{r pathlist, eval=TRUE} 
## Map to pathways (only 100 pathways in order to reduce the runtime)
pathlistDBsub <- pathlistDB[1:100]
pathlist <- omics2pathlist(data=studyData, pathlistDBsub, featureAnno, 
                           restrictUp=100, restrictDown=20, minPathSize=10) 
``` 

# BioMM framework

## Recapitulation
Briefly, the BioMM framework consists of two learning stages [1]. During the 
first stage, biological meta-information is used to 'compress' the variables 
of the original dataset into pathway-level 'latent variables' (henceforth 
called stage-2 data) using either supervised or unsupervised learning models. 
In the second stage, a supervised model is built using the stage-2 data with 
non-negative outcome-associated features for prediction. 

## End-to-end prediction modules
### Interface to machine learning models 
The end-to-end prediction is performed using `BioMM()` function. Both 
supervised and unsupervised learning are implemented in the BioMM framework, 
which are indicated by the argument `supervisedStage1=TRUE` or 
`supervisedStage1=FALSE`. Commonly used supervised classifiers: generalized 
regression models with lasso, ridge or elastic net regularization (GLM) [4], 
support vector machine (SVM) [3] and random forest [2] are included. For the 
unsupervised method, regular or sparse constrained principal component 
analysis (PCA) [5] is used. Generic resampling methods include 
cross-validation (CV) and bootstrapping (BS) procedures as the argument 
`resample1="CV"` or `resample1="BS"`. Stage-2 data is reconstructed using 
either resampling methods during machine learning prediction or independent 
test set prediction if the argument `testData` is provided.

#### Example  
To apply random forest model, we use the argument `classifier=randForest`
in `BioMM()` with the classification mode at both 
stages. `predMode` indicate the prediction type, here we use 
classification for binary outcome prediction. A set of model hyper-parameters 
are supplied by the argument `paramlist` at both stages. 
Pathway-based stratification is carried out in this example. We 
focused on the autosomal region to limit the potential influence of sex on 
machine learning due to the phenomenon of X chromosome inactivation or the 
existence of an additional X chromosome in female samples. Therefore it's 
suggested to exclude sex chromosome in the user-supplied `featureAnno` input 
file. 

```{r BioMMrandForest, eval=TRUE}
## Parameters
supervisedStage1=TRUE
classifier <- "randForest"
predMode <- "classification"
paramlist <- list(ntree=300, nthreads=10)   
param1 <- MulticoreParam(workers = 1)
param2 <- MulticoreParam(workers = 10)
## If BioMM is installed from Github, please use the following assignment:
## param1 <- 1
## param2 <- 10

studyDataSub <- studyData[,1:2000] ## to reduce the runtime
set.seed(123)
result <- BioMM(trainData=studyDataSub, testData=NULL, pathlistDB, featureAnno, 
                restrictUp=100, restrictDown=10, minPathSize=10, 
                supervisedStage1, typePCA="regular", 
                resample1="BS", resample2="CV", dataMode="allTrain",
                repeatA1=50, repeatA2=1, repeatB1=20, repeatB2=1, 
                nfolds=10, FSmethod1=NULL, FSmethod2=NULL, 
                cutP1=0.1, cutP2=0.1, fdr2=NULL, FScore=param1, 
                classifier, predMode, 
                paramlist, innerCore=param2)
print(result)
``` 

Other machine learning models can be employed with the following respective 
parameter settings. For the classifier `"SVM"`, parameters can be tuned using 
an internal cross validation if `tuneP=TRUE`. For generalized regression model 
`glmnet`, elastic net is specified by the input argument `alpha=0.5`. 
Alternatively, `alpha=1` is for the lasso and `alpha=0` is the ridge. For the 
unsupervised learning `supervisedStage1=FALSE`, regular PCA 
`typePCA="regular"` is applied and followed with random forest classification 
`classifier2=TRUE`.

```{r BioMMpara, eval=TRUE}
## SVM 
supervisedStage1=TRUE
classifier <- "SVM"
predMode <- "classification"
paramlist <- list(tuneP=FALSE, kernel="radial", 
                  gamma=10^(-3:-1), cost=10^(-3:1))

## GLM with elastic-net
supervisedStage1=TRUE
classifier <- "glmnet"
predMode <- "classification" 
paramlist <- list(family="binomial", alpha=0.5, 
                   ypeMeasure="mse", typePred="class")

## PCA + random forest
supervisedStage1=FALSE
classifier <- "randForest"
predMode <- "classification"
paramlist <- list(ntree=300, nthreads=10)  
```


### Interface to biological stratification strategies
For stratification of predictors using biological information, various 
strategies can be applied. Currently, `BioMM()` integrates gene 
ontological pathway based stratification, which not only accounts for epistasis 
between first level variables within the functional category, but also considers
the interaction between pathways. Therefore, this may provide better information on functional insight.  

#### Example
End-to-end prediction based on pathway-wide stratification on genome-wide DNA 
methylation data is demonstrated below. PCA is used at stage-1 to reconstruct 
pathway level data, then the random forest model with 10-fold cross validation
is applied on stage-2 data to estimate the prediction performance.

```{r BioMMpathway, eval=TRUE}
## Parameters
supervisedStage1=FALSE
classifier <- "randForest"
predMode <- "classification"
paramlist <- list(ntree=300, nthreads=10)   
param1 <- MulticoreParam(workers = 1)
param2 <- MulticoreParam(workers = 10)
## If BioMM is installed from Github, please use the following assignment:
## param1 <- 1
## param2 <- 10

set.seed(123)
result <- BioMM(trainData=studyData, testData=NULL, 
                pathlistDBsub, featureAnno, 
                restrictUp=100, restrictDown=10, minPathSize=10, 
                supervisedStage1, typePCA="regular", 
                resample1="BS", resample2="CV", dataMode="allTrain",
                repeatA1=40, repeatA2=1, repeatB1=40, repeatB2=1, 
                nfolds=10, FSmethod1=NULL, FSmethod2=NULL, 
                cutP1=0.1, cutP2=0.1, fdr2=NULL, FScore=param1, 
                classifier, predMode, paramlist, innerCore=param2)
print(result)
``` 

## Stage-2 data exploration 
### Generation of stage-2 data
Here we demonstrate using supervised random forest method on genome-wide DNA 
methylation. Gene ontological pathways are used for the generation of stage-2 
data.

```{r stage2data, eval=TRUE}
## Pathway level data or stage-2 data prepared by reconBySupervised()
stage2dataA <- readRDS(system.file("extdata", "/stage2dataA.rds", 
                       package="BioMM"))

head(stage2dataA[,1:5])
dim(stage2dataA)
``` 

```{r stage2dataAprepare, eval=FALSE} 
#### Alternatively, 'stage2dataA' can be created by the following code:
## Parameters  
classifier <- "randForest" 
predMode <- "probability" 
paramlist <- list(ntree=300, nthreads=40)  
param1 <- MulticoreParam(workers = 1)
param2 <- MulticoreParam(workers = 10) 
## If BioMM is installed from Github, please use the following assignment:
## param1 <- 1
## param2 <- 10
set.seed(123)
## This will take a bit longer to run
stage2dataA <- reconBySupervised(trainDataList=pathlist, testDataList=NULL,
                            resample="BS", dataMode="allTrain",
                            repeatA=25, repeatB=1, nfolds=10,
                            FSmethod=NULL, cutP=0.1, fdr=NULL, FScore=param1,
                            classifier, predMode, paramlist,
                            innerCore=param2, outFileA=NULL, outFileB=NULL)

``` 

### Visualization
#### Explained variation of stage-2 data
The distribution of the proportion of variance explained for the individual 
generated feature of stage-2 data for the classification task is illustrated 
`plotVarExplained()` below. Nagelkerke pseudo R-squared measure is used to 
compute the explained variance. The argument `posF=TRUE` indicates that only 
positively outcome-associated features are plotted, since negative 
associations likely reflect random effects in the underlying data [6].

``` {r stage2dataViz, eval=TRUE}
param <- MulticoreParam(workers = 1) 
## If BioMM is installed from Github, please use the following assignment:
## param <- 1 
plotVarExplained(data=stage2dataA, posF=TRUE, 
                 core=param, horizontal=FALSE, fileName=NULL)
``` 
![](R2explained.png)

#### Prioritization of outcome-associated functional patterns 
`plotRankedFeature()` is employed to rank and visualize the outcome-associated 
features from stage-2 data. The argument `topF=10` and `posF=TRUE` are used to 
define the top 10 positively outcome-associated features. Nagelkerke pseudo 
R-squared measure is utilized to evaluate the importance of the ranked 
features as indicated by the argument `rankMetric="R2"`. The size of the 
investigated pathway is pictured as the argument `colorMetric="size"`. 

``` {r topFstage2data, eval=TRUE} 
param <- MulticoreParam(workers = 10) 
## If BioMM is installed from Github, please use the following assignment:
## param <- 1 
topPath <- plotRankedFeature(data=stage2dataA, 
                             posF=TRUE, topF=10, 
                             blocklist=pathlist,
                             rankMetric="R2", 
                             colorMetric="size", 
                             core=param, fileName=NULL)
``` 
![](plotTopF10_R2_pathway.png)

#### The significance of CpGs in pathways of interest
`cirPlot4pathway()` illustrates the significance of the individual CpGs falling 
into a set of pathways. Here the top 10 outcome-associated pathways are investigated.
Negative log P value is used to define the significance of each CpG within these pathways.

``` {r cirPlot, eval=TRUE}  
core <- MulticoreParam(workers = 10)  
## If BioMM is installed from Github, use the following assignment:
## core <- 10
pathID <- gsub("\\.", ":", rownames(topPath))
pathSet <- pathlist[is.element(names(pathlist), pathID)]
pathMatch <- pathSet[match(pathID, names(pathSet))]

cirPlot4pathway(datalist=pathMatch, 
                topPathID=names(pathMatch), core, fileName=NULL)


``` 
![](Circular-Manhattan.pval.jpg)

## Computational consideration
BioMM with supervised models at both stages incorporating pathway based 
stratification method will take longer to run than unsupervised
approaches. But the prediction is more powerful. Therefore, we suggest the former even
if the computation is more demanding, as the adoption of 5G is pushing advances 
in computational storage and speed. Parallel computing is implemented and 
recommended for such scenario. In this vignette, due to the runtime, we 
only showcased the smaller examples and models with less computation. 


# Session information

``` {r sessioninfo, eval=TRUE} 
sessionInfo()
```  

# References

[1] NIPS ML4H submission: Chen, J. and Schwarz, E., 2017. BioMM: 
Biologically-informed Multi-stage Machine learning for identification of 
epigenetic fingerprints. arXiv preprint arXiv:1712.00336. 

[2] Breiman, L. (2001). "Random forests." Machine learning 45(1): 5-32.

[3] Cortes, C., & Vapnik, V. (1995). "Support-vector networks." 
Machine learning 20(3): 273-297.

[4] Friedman, J., Hastie, T., & Tibshirani, R. (2010). "Regularization paths 
for generalized linear models via coordinate descent." 
Journal of statistical software 33(1): 1.

[5] Wold, S., Esbensen, K., & Geladi, P. (1987). "Principal component 
analysis." Chemometrics and intelligent laboratory systems 2(1-3): 37-52.

[6] Claudia Perlich and Grzegorz Swirszcz. On cross-validation and stacking: 
Building seemingly predictive models on random data. ACM SIGKDD Explorations 
Newsletter, 12(2):11-15, 2011. 

# Questions & Comments
If you have any questions or comments ? 

Contact: junfang.chen@zi-mannheim.de





