---
title: "_systemPipeR_: NGS workflow and report generation environment" 
author: "Author: Thomas Girke (thomas.girke@ucr.edu)"
date: "Last update: `r format(Sys.time(), '%d %B, %Y')`" 
output:
  BiocStyle::html_document:
    toc: true
    toc_depth: 3
    fig_caption: yes

fontsize: 14pt
bibliography: bibtex.bib
---
<!--
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%% \VignetteKeywords{compute cluster, pipeline, reports}
%% \VignettePackage{systemPipeR}
-->

<!---
- Compile from command-line
echo "rmarkdown::render('systemPipeR.Rmd')" | R -slave; R CMD Stangle systemPipeR.Rmd

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git commit -am "some edits"; git push -u origin master

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```{r style, echo = FALSE, results = 'asis'}
BiocStyle::markdown()
options(width=100, max.print=1000)
knitr::opts_chunk$set(
    eval=as.logical(Sys.getenv("KNITR_EVAL", "TRUE")),
    cache=as.logical(Sys.getenv("KNITR_CACHE", "TRUE")))
```

```{r setup, echo=FALSE, messages=FALSE, warnings=FALSE}
suppressPackageStartupMessages({
    library(systemPipeR)
    library(BiocParallel)
    library(Biostrings)
    library(Rsamtools)
    library(GenomicRanges)
    library(ggplot2)
    library(GenomicAlignments)
    library(ShortRead)
    library(ape)
})
```

Note: the most recent version of this tutorial can be found <a href="https://htmlpreview.github.io/?https://github.com/tgirke/systemPipeR/blob/master/vignettes/systemPipeR.html">here</a> and a short overview slide show [here](https://htmlpreview.github.io/?https://github.com/tgirke/systemPipeR/master/inst/extdata/slides/systemPipeRslides.html).

# Introduction

[_`systemPipeR`_](http://www.bioconductor.org/packages/devel/bioc/html/systemPipeR.html) provides utilities for building and running automated end-to-end analysis workflows for a wide range of next generation sequence (NGS) applications such as RNA-Seq, ChIP-Seq, VAR-Seq and Ribo-Seq [@Girke2014-oy]. Important features include a uniform workflow interface across different NGS applications, automated report generation, and support for running both R and command-line software, such as NGS aligners or peak/variant callers, on local computers or compute clusters. The latter supports interactive job submissions and batch submissions to queuing systems of clusters. For instance, _`systemPipeR`_ can be used with most command-line aligners such as `BWA` [@Li2013-oy; @Li2009-oc], `TopHat2` [@Kim2013-vg] and `Bowtie2` [@Langmead2012-bs], as well as the R-based NGS aligners [_`Rsubread`_](http://www.bioconductor.org/packages/devel/bioc/html/Rsubread.html) [@Liao2013-bn] and [_`gsnap (gmapR)`_](http://www.bioconductor.org/packages/devel/bioc/html/gmapR.html) [@Wu2010-iq]. Efficient handling of complex sample sets (_e.g._ FASTQ/BAM files) and experimental designs is facilitated by a well-defined sample annotation infrastructure which improves reproducibility and user-friendliness of many typical analysis workflows in the NGS area [@Lawrence2013-kt]. 

Motivation and advantages of _`sytemPipeR`_ environment:

1. Facilitates design of complex NGS workflows involving multiple R/Bioconductor packages
2. Common workflow interface for different NGS applications
3. Makes NGS analysis with Bioconductor utilities more accessible to new users
4. Simplifies usage of command-line software from within R
5. Reduces complexity of using compute clusters for R and command-line software
6. Accelerates runtime of workflows via parallelzation on computer systems with mutiple CPU cores and/or multiple compute nodes
6. Automates generation of analysis reports to improve reproducibility

A central concept for designing workflows within the _`sytemPipeR`_ environment is the use of workflow management containers called _`SYSargs`_ (see Figure 1). Instances of this S4 object class are constructed by the _`systemArgs`_ function from two simple tabular files: a _`targets`_ file and a _`param`_ file. The latter is optional for workflow steps lacking command-line software. Typically, a _`SYSargs`_ instance stores all sample-level inputs as well as the paths to the corresponding outputs generated by command-line- or R-based software generating sample-level output files, such as read preprocessors (trimmed/filtered FASTQ files), aligners (SAM/BAM files), variant callers (VCF/BCF files) or peak callers (BED/WIG files). Each sample level input/outfile operation uses its own _`SYSargs`_ instance. The outpaths of _`SYSargs`_ usually define the sample inputs for the next _`SYSargs`_ instance. This connectivity is established by writing the outpaths with the _`writeTargetsout`_ function to a new _`targets`_ file that serves as input to the next _`systemArgs`_ call. Typically, the user has to provide only the initial _`targets`_ file. All downstream _`targets`_ files are generated automatically. By chaining several _`SYSargs`_ steps together one can construct complex workflows involving many sample-level input/output file operations with any combinaton of command-line or R-based software. 

![](SystemPipeR_Workflow.png)
<div align="center">**Figure 1:** Workflow design structure of *systemPipeR* </div></br>

The intended way of running _`sytemPipeR`_ workflows is via _`*.Rnw`_ or _`*.Rmd`_ files, which can be executed either line-wise in interactive mode or with a single command from R or the command-line using a [_`Makefile`_](https://github.com/tgirke/systemPipeR/blob/master/inst/extdata/Makefile). This way comprehensive and reproducible analysis reports in PDF or HTML format can be generated in a fully automated manner by making use of the highly functional reporting utilities available for R. Templates for setting up custom project reports are provided as _`*.Rnw`_ files by the helper package _`systemPipeRdata`_ and in the vignettes subdirectory of _`systemPipeR`_. The corresponding PDFs of these report templates are available here: [_`systemPipeRNAseq`_](https://github.com/tgirke/systemPipeR/blob/master/vignettes/systemPipeRNAseq.pdf?raw=true), [_`systemPipeRIBOseq`_](https://github.com/tgirke/systemPipeR/blob/master/vignettes/systemPipeRIBOseq.pdf?raw=true), [_`systemPipeChIPseq`_](https://github.com/tgirke/systemPipeR/blob/master/vignettes/systemPipeChIPseq.pdf?raw=true) and [_`systemPipeVARseq`_](https://github.com/tgirke/systemPipeR/blob/master/vignettes/systemPipeVARseq.pdf?raw=true). To work with _`*.Rnw`_ or _`*.Rmd`_ files efficiently, basic knowledge of [_`Sweave`_](https://www.stat.uni-muenchen.de/~leisch/Sweave/) or [_`knitr`_](http://yihui.name/knitr/) and [_`Latex`_](http://www.latex-project.org/) or [_`R Markdown v2`_](http://rmarkdown.rstudio.com/) is required. 

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# Getting Started

## Installation

The R software for running [_`systemPipeR`_](http://www.bioconductor.org/packages/devel/bioc/html/systemPipeR.html) can be downloaded from [_CRAN_](http://cran.at.r-project.org/). The _`systemPipeR`_ environment can be installed from R using the _`biocLite`_ install command. The associated data package [_`systemPipeRdata`_](https://github.com/tgirke/systemPipeRdata) can be used to generate _`systemPipeR`_ workflow environments with a single command ([see below](#load-sample-data-and-workflow-templates)) containing all parameter files and sample data required to quickly test and run workflows. 

    
```{r install, eval=FALSE}
source("http://bioconductor.org/biocLite.R") # Sources the biocLite.R installation script 
biocLite("systemPipeR") # Installs systemPipeR 
biocLite("systemPipeRdata") # Installs systemPipeRdata
```
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## Loading package and documentation

```{r documentation, eval=FALSE}
library("systemPipeR") # Loads the package
library(help="systemPipeR") # Lists package info
vignette("systemPipeR") # Opens vignette
```
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## Load sample data and workflow templates
The mini sample FASTQ files used by this overview vignette as well as the associated workflow reporting vignettes can be loaded via the _`systemPipeRdata`_ package as shown below. The chosen data set [`SRP010938`](http://www.ncbi.nlm.nih.gov/sra/?term=SRP010938) contains 18 paired-end (PE) read sets from _Arabidposis thaliana_ [@Howard2013-fq]. To minimize processing time during testing, each FASTQ file has been subsetted to 90,000-100,000 randomly sampled PE reads that map to the first 100,000 nucleotides of each chromosome of the _A. thalina_ genome. The corresponding reference genome sequence (FASTA) and its GFF annotion files (provided in the same download) have been truncated accordingly. This way the entire test sample data set requires less than 200MB disk storage space. A PE read set has been chosen for this test data set for flexibility, because it can be used for testing both types of analysis routines requiring either SE (single end) reads or PE reads. 

The following loads one of the available NGS workflow templates (here RNA-Seq) into the user's current working directory. At the moment, the package includes workflow templates for RNA-Seq, ChIP-Seq, VAR-Seq and Ribo-Seq. Templates for additional NGS applications will be provided in the future.
```{r genRna_workflow, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow="rnaseq")
setwd("rnaseq")
```

The working environment of the sample data loaded in previous step contains the following preconfigured directory structure: 

* <span style="color:grey">_**workflow/**_</span> 
    + This is the directory of the R session running the workflow.
    + Run script ( _\*.Rnw_ or _\*.Rmd_) and sample annotation (_targets.txt_) files are located here.
    + Note, this directory can have any name (_e.g._ <span style="color:grey">_**rnaseq**_</span>, <span style="color:grey">_**varseq**_</span>). Changing its name does not require any modifications in the run script(s).
    + Important subdirectories: 
        + <span style="color:grey">_**param/**_</span> 
            + Stores parameter files such as: _\*.param_, _\*.tmpl_ and _\*\_run.sh_.
        + <span style="color:grey">_**data/**_ </span>
            + FASTQ samples 
            + Reference FASTA file
            + Annotations
            + etc.
        + <span style="color:grey">_**results/**_</span>
            + Alignment, variant and peak files (BAM, VCF, BED) 
            + Tabular result files
            + Images and plots
            + etc.

The sample workflows provided by the package are based on the above directory structure, where directory names are indicated in  <span style="color:grey">_**grey**_</span>. 
Users can change this structure as needed, but need to adjust the code in their workflows accordingly. 

The following parameter files are included in each workflow template: 

1. _`targets.txt`_: initial one provided by user; downstream _`targets_*.txt`_ files are generated automatically
2. _`*.param`_: defines parameter for input/output file operations, _e.g._ _`trim.param`_, _`bwa.param`_, _`vartools.parm`_, ...
3. _`*_run.sh`_: optional bash script, _e.g._: _`gatk_run.sh`_
4. Compute cluster environment (skip on single machine):
    + _`.BatchJobs`_: defines type of scheduler for _`BatchJobs`_
    + _`*.tmpl`_: specifies parameters of scheduler used by a system, _e.g._ Torque, SGE, StarCluster, Slurm, etc.

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## Structure of _`targets`_ file

The _`targets`_ file defines all input files (_e.g._ FASTQ, BAM, BCF) and sample comparisons of an analysis workflow. The following shows the format of a sample _`targets`_ file included in the package. It also can be viewed and downloaded from _`systemPipeR`_'s GitHub repository [here](https://github.com/tgirke/systemPipeR/blob/master/inst/extdata/targets.txt). In a target file with a single type of input files, here FASTQ files of single end (SE) reads, the first three columns are mandatory including their column names, while it is four mandatory columns for FASTQ files of PE reads. All subsequent columns are optional and any number of additional columns can be added as needed.

### Structure of _`targets`_ file for single end (SE) samples
```{r targetsSE, eval=TRUE}
library(systemPipeR)
targetspath <- system.file("extdata", "targets.txt", package="systemPipeR") 
read.delim(targetspath, comment.char = "#")
```
To work with custom data, users need to generate a _`targets`_ file containing the paths to their own FASTQ files and then provide under _`targetspath`_ the path to the corresponding _`targets`_ file. 

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### Structure of _`targets`_ file for paired end (PE) samples

```{r targetsPE, eval=TRUE}
targetspath <- system.file("extdata", "targetsPE.txt", package="systemPipeR")
read.delim(targetspath, comment.char = "#")[1:2,1:6]
```
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### Sample comparisons
Sample comparisons are defined in the header lines of the _`targets`_ file starting with '``# <CMP>``'. 

```{r comment_lines, eval=TRUE}
readLines(targetspath)[1:4]
```

The function _`readComp`_ imports the comparison information and stores it in a _`list`_. Alternatively, _`readComp`_ can obtain the comparison information from the corresponding _`SYSargs`_ object (see below). 
Note, these header lines are optional. They are mainly useful for controlling comparative analyses according to certain biological expectations, such as identifying differentially expressed genes in RNA-Seq experiments based on simple pair-wise comparisons.
 
```{r targetscomp, eval=TRUE}
readComp(file=targetspath, format="vector", delim="-")
```
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## Structure of _`param`_ file and _`SYSargs`_ container
The _`param`_ file defines the parameters of a chosen command-line software. The following shows the format of a sample _`param`_ file provided by this package. 

```{r param_structure, eval=TRUE}
parampath <- system.file("extdata", "tophat.param", package="systemPipeR")
read.delim(parampath, comment.char = "#")
```

The _`systemArgs`_ function imports the definitions of both the _`param`_ file and the _`targets`_ file, and stores all relevant information in a _`SYSargs`_ S4 class object. To run the pipeline without command-line software, one can assign _`NULL`_ to _`sysma`_ instead of a _`param`_ file. In addition, one can start the _`systemPipeR`_ workflow with pre-generated BAM files by providing a targets file where the _`FileName`_ column gives the paths to the BAM files and _`sysma`_ is assigned _`NULL`_.

```{r param_import, eval=TRUE}
args <- suppressWarnings(systemArgs(sysma=parampath, mytargets=targetspath))
args
```

Several accessor functions are available that are named after the slot names of the _`SYSargs`_ object.
```{r sysarg_access, eval=TRUE}
names(args)
modules(args)
cores(args)
outpaths(args)[1]
sysargs(args)[1]
```

The content of the _`param`_ file can also be returned as JSON object as follows (requires _`rjson`_ package).
```{r sysarg_json, eval=TRUE}
systemArgs(sysma=parampath, mytargets=targetspath, type="json")
```
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# Workflow overview

## Define environment settings and samples

Load packages and generate workflow environment (here for RNA-Seq)
```{r load_package, eval=FALSE}
library(systemPipeR)
library(systemPipeRdata)
genWorkenvir(workflow="rnaseq")
setwd("rnaseq")
```

Construct _`SYSargs`_ object from _`param`_ and _`targets`_ files.
```{r construct_sysargs, eval=FALSE}
args <- systemArgs(sysma="param/trim.param", mytargets="targets.txt")
```
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## Read Preprocessing
The function _`preprocessReads`_ allows to apply predefined or custom
read preprocessing functions to all FASTQ files referenced in a
_`SYSargs`_ container, such as quality filtering or adaptor trimming
routines. The paths to the resulting output FASTQ files are stored in the
_`outpaths`_ slot of the _`SYSargs`_ object. Internally,
_`preprocessReads`_ uses the _`FastqStreamer`_ function from
the _`ShortRead`_ package to stream through large FASTQ files in a
memory-efficient manner. The following example performs adaptor trimming with
the _`trimLRPatterns`_ function from the _`Biostrings`_ package.
After the trimming step a new targets file is generated (here
_`targets_trim.txt`_) containing the paths to the trimmed FASTQ files.
The new targets file can be used for the next workflow step with an updated
_`SYSargs`_ instance, \textit{e.g.} running the NGS alignments using the
trimmed FASTQ files.
 
```{r preprocessing, eval=FALSE}
preprocessReads(args=args, Fct="trimLRPatterns(Rpattern='GCCCGGGTAA', subject=fq)", 
                batchsize=100000, overwrite=TRUE, compress=TRUE)
writeTargetsout(x=args, file="targets_trim.txt")
```

The following example shows how one can design a custom read preprocessing function 
using utilities provided by the _`ShortRead`_ package, and then run it
in batch mode with the _'preprocessReads'_ function (here on paired-end reads).
```{r custom_preprocessing, eval=FALSE}
args <- systemArgs(sysma="param/trimPE.param", mytargets="targetsPE.txt")
filterFct <- function(fq, cutoff=20, Nexceptions=0) {
    qcount <- rowSums(as(quality(fq), "matrix") <= cutoff)
    fq[qcount <= Nexceptions] # Retains reads where Phred scores are >= cutoff with N exceptions
}
preprocessReads(args=args, Fct="filterFct(fq, cutoff=20, Nexceptions=0)", batchsize=100000)
writeTargetsout(x=args, file="targets_PEtrim.txt")
```
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## FASTQ quality report
The following _`seeFastq`_ and _`seeFastqPlot`_ functions generate and plot a series of
useful quality statistics for a set of FASTQ files including per cycle quality
box plots, base proportions, base-level quality trends, relative k-mer
diversity, length and occurrence distribution of reads, number of reads above
quality cutoffs and mean quality distribution.  
```{r fastq_quality, eval=FALSE}
fqlist <- seeFastq(fastq=infile1(args), batchsize=10000, klength=8)
pdf("./results/fastqReport.pdf", height=18, width=4*length(fqlist))
seeFastqPlot(fqlist)
dev.off()
```
![](fastqReport.png)
<div align="center">**Figure 2:** FASTQ quality report </div></br>

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Parallelization of QC report on single machine with multiple cores

```{r fastq_quality_parallel_single, eval=FALSE}
args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt")
f <- function(x) seeFastq(fastq=infile1(args)[x], batchsize=100000, klength=8)
fqlist <- bplapply(seq(along=args), f, BPPARAM = MulticoreParam(workers=8))
seeFastqPlot(unlist(fqlist, recursive=FALSE))
```
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Parallelization of QC report via scheduler (_e.g._ Torque) across several compute nodes

```{r fastq_quality_parallel_cluster, eval=FALSE}
library(BiocParallel); library(BatchJobs)
f <- function(x) {
    library(systemPipeR)
    args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt")
    seeFastq(fastq=infile1(args)[x], batchsize=100000, klength=8)
}
funs <- makeClusterFunctionsTorque("torque.tmpl")
param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs)
register(param)
fqlist <- bplapply(seq(along=args), f)
seeFastqPlot(unlist(fqlist, recursive=FALSE))
```
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## Alignment with _`Tophat2`_
Build _`Bowtie2`_ index.

```{r bowtie_index, eval=FALSE}
args <- systemArgs(sysma="param/tophat.param", mytargets="targets.txt")
moduleload(modules(args)) # Skip if module system is not available
system("bowtie2-build ./data/tair10.fasta ./data/tair10.fasta")
```

Execute _`SYSargs`_ on a single machine without submitting to a queuing system of a compute cluster. This way the input FASTQ files will be processed sequentially. If available, multiple CPU cores can be used for processing each file. The number of CPU cores (here 4) to use for each process is defined in the _`*.param`_ file. With _`cores(args)`_ one can return this value from the _`SYSargs`_ object. Note, if a module system is not installed or used, then the corresponding _`*.param`_ file needs to be edited accordingly by either providing an empty field in the line(s) starting with _`module`_ or by deleting these lines.

```{r run_bowtie_single, eval=FALSE}
bampaths <- runCommandline(args=args)
```

Alternatively, the computation can be greatly accelerated by processing many files in parallel using several compute nodes of a cluster, where a scheduling/queuing system is used for load balancing. To avoid over-subscription of CPU cores on the compute nodes, the value from _`cores(args)`_ is passed on to the submission command, here _`nodes`_ in the _`resources`_ list object. The number of independent parallel cluster processes is defined under the _`Njobs`_ argument. The following example will run 18 processes in parallel using for each 4 CPU cores. If the resources available on a cluster allow to run all 18 processes at the same time then the shown sample submission will utilize in total 72 CPU cores. Note, _`clusterRun`_ can be used with most queueing systems as it is based on utilities from the _`BatchJobs`_ package which supports the use of template files (_`*.tmpl`_) for defining the run parameters of different schedulers. To run the following code, one needs to have both a conf file (see _`.BatchJob`_ samples [here](https://code.google.com/p/batchjobs/wiki/DortmundUsage)) and a template file (see _`*.tmpl`_ samples [here](https://github.com/tudo-r/BatchJobs/tree/master/examples)) for the queueing available on a system. The following example uses the sample conf and template files for the Torque scheduler provided by this package.  

```{r run_bowtie_parallel, eval=FALSE}
resources <- list(walltime="20:00:00", nodes=paste0("1:ppn=", cores(args)), memory="10gb")
reg <- clusterRun(args, conffile=".BatchJobs.R", template="torque.tmpl", Njobs=18, runid="01", 
                  resourceList=resources)
waitForJobs(reg)
```

Useful commands for monitoring progress of submitted jobs
```{r process_monitoring, eval=FALSE}
showStatus(reg)
file.exists(outpaths(args))
sapply(1:length(args), function(x) loadResult(reg, x)) # Works after job completion
```
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## Read and alignment count stats
Generate table of read and alignment counts for all samples. 
```{r align_stats1, eval=FALSE}
read_statsDF <- alignStats(args) 
write.table(read_statsDF, "results/alignStats.xls", row.names=FALSE, quote=FALSE, sep="\t")
```

The following shows the first four lines of the sample alignment stats file provided by the _`systemPipeR`_ package. For simplicity the number of PE reads is multiplied here by 2 to approximate proper alignment frequencies where each read in a pair is counted. 
```{r align_stats2, eval=TRUE}
read.table(system.file("extdata", "alignStats.xls", package="systemPipeR"), header=TRUE)[1:4,]
```

Parallelization of read/alignment stats on single machine with multiple cores
```{r align_stats_parallel, eval=FALSE}
f <- function(x) alignStats(args[x])
read_statsList <- bplapply(seq(along=args), f, BPPARAM = MulticoreParam(workers=8))
read_statsDF <- do.call("rbind", read_statsList)
```

Parallelization of read/alignment stats via scheduler (_e.g._ Torque) across several compute nodes 
```{r align_stats_parallel_cluster, eval=FALSE}
library(BiocParallel); library(BatchJobs)
f <- function(x) {
    library(systemPipeR)
    args <- systemArgs(sysma="tophat.param", mytargets="targets.txt")
    alignStats(args[x])
}
funs <- makeClusterFunctionsTorque("torque.tmpl")
param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs)
register(param)
read_statsList <- bplapply(seq(along=args), f)
read_statsDF <- do.call("rbind", read_statsList)
```
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## Create symbolic links for viewing BAM files in IGV
The genome browser IGV supports reading of indexed/sorted BAM files via web URLs. This way it can be avoided to create unnecessary copies of these large files. To enable this approach, an HTML directory with http access needs to be available in the user account (_e.g._ _`home/publichtml`_) of a system. If this is not the case then the BAM files need to be moved or copied to the system where IGV runs. In the following, _`htmldir`_ defines the path to the HTML directory with http access where the symbolic links to the BAM files will be stored. The corresponding URLs will be written to a text file specified under the `_urlfile`_ argument. 
```{r igv, eval=FALSE}
symLink2bam(sysargs=args, htmldir=c("~/.html/", "somedir/"), 
            urlbase="http://myserver.edu/~username/", 
        urlfile="IGVurl.txt")
```
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## Alternative NGS Aligners

### Alignment with _`Bowtie2`_ (_e.g._ for miRNA profiling)
The following example runs _`Bowtie2`_ as a single process without submitting it to a cluster.
```{r bowtie2, eval=FALSE}
args <- systemArgs(sysma="bowtieSE.param", mytargets="targets.txt")
moduleload(modules(args)) # Skip if module system is not available
bampaths <- runCommandline(args=args)
```

Alternatively, submit the job to compute nodes.
```{r bowtie2_cluster, eval=FALSE}
resources <- list(walltime="20:00:00", nodes=paste0("1:ppn=", cores(args)), memory="10gb")
reg <- clusterRun(args, conffile=".BatchJobs.R", template="torque.tmpl", Njobs=18, runid="01", 
                  resourceList=resources)
waitForJobs(reg)
```
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### Alignment with _`BWA-MEM`_ (_e.g._ for VAR-Seq)
The following example runs BWA-MEM as a single process without submitting it to a cluster.
```{r bwamem_cluster, eval=FALSE}
args <- systemArgs(sysma="param/bwa.param", mytargets="targets.txt")
moduleload(modules(args)) # Skip if module system is not available
system("bwa index -a bwtsw ./data/tair10.fasta") # Indexes reference genome
bampaths <- runCommandline(args=args[1:2])
```
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### Alignment with _`Rsubread`_ (_e.g._ for RNA-Seq)
The following example shows how one can use within the \Rpackage{systemPipeR} environment the R-based 
aligner \Rpackage{Rsubread} or other R-based functions that read from input files and write to output files.
```{r rsubread, eval=FALSE}
library(Rsubread)
args <- systemArgs(sysma="param/rsubread.param", mytargets="targets.txt")
buildindex(basename=reference(args), reference=reference(args)) # Build indexed reference genome
align(index=reference(args), readfile1=infile1(args)[1:4], input_format="FASTQ", 
      output_file=outfile1(args)[1:4], output_format="SAM", nthreads=8, indels=1, TH1=2)
for(i in seq(along=outfile1(args))) asBam(file=outfile1(args)[i], destination=gsub(".sam", "", outfile1(args)[i]), overwrite=TRUE, indexDestination=TRUE)
```
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### Alignment with _`gsnap`_ (_e.g._ for VAR-Seq and RNA-Seq)
Another R-based short read aligner is _`gsnap`_ from the _`gmapR`_ package [@Wu2010-iq]. 
The code sample below introduces how to run this aligner on multiple nodes of a compute cluster. 
```{r gsnap, eval=FALSE}
library(gmapR); library(BiocParallel); library(BatchJobs)
args <- systemArgs(sysma="param/gsnap.param", mytargets="targetsPE.txt")
gmapGenome <- GmapGenome(reference(args), directory="data", name="gmap_tair10chr/", create=TRUE)
f <- function(x) {
    library(gmapR); library(systemPipeR)
    args <- systemArgs(sysma="gsnap.param", mytargets="targetsPE.txt")
    gmapGenome <- GmapGenome(reference(args), directory="data", name="gmap_tair10chr/", create=FALSE)
    p <- GsnapParam(genome=gmapGenome, unique_only=TRUE, molecule="DNA", max_mismatches=3)
    o <- gsnap(input_a=infile1(args)[x], input_b=infile2(args)[x], params=p, output=outfile1(args)[x])
}
funs <- makeClusterFunctionsTorque("torque.tmpl")
param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs)
register(param)
d <- bplapply(seq(along=args), f)
```
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## Read counting for mRNA profiling experiments
Create _`txdb`_ (needs to be done only once)
```{r create_txdb, eval=FALSE}
library(GenomicFeatures)
txdb <- makeTxDbFromGFF(file="data/tair10.gff", format="gff", dataSource="TAIR", organism="A. thaliana")
saveDb(txdb, file="./data/tair10.sqlite")
```
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The following performs read counting with _`summarizeOverlaps`_ in parallel mode with multiple cores. 
```{r read_counting_multicore, eval=FALSE}
library(BiocParallel)
txdb <- loadDb("./data/tair10.sqlite")
eByg <- exonsBy(txdb, by="gene")
bfl <- BamFileList(outpaths(args), yieldSize=50000, index=character())
multicoreParam <- MulticoreParam(workers=4); register(multicoreParam); registered()
counteByg <- bplapply(bfl, function(x) summarizeOverlaps(eByg, x, mode="Union", ignore.strand=TRUE, inter.feature=TRUE, singleEnd=TRUE)) # Note: for strand-specific RNA-Seq set 'ignore.strand=FALSE' and for PE data set 'singleEnd=FALSE'
countDFeByg <- sapply(seq(along=counteByg), function(x) assays(counteByg[[x]])$counts)
rownames(countDFeByg) <- names(rowRanges(counteByg[[1]])); colnames(countDFeByg) <- names(bfl)
rpkmDFeByg <- apply(countDFeByg, 2, function(x) returnRPKM(counts=x, ranges=eByg))
write.table(countDFeByg, "results/countDFeByg.xls", col.names=NA, quote=FALSE, sep="\t")
write.table(rpkmDFeByg, "results/rpkmDFeByg.xls", col.names=NA, quote=FALSE, sep="\t")
```
Please note, in addition to read counts this step generates RPKM normalized expression values. For most statistical differential expression or abundance analysis methods, such as _`edgeR`_ or _`DESeq2`_, the raw count values should be used as input. The usage of RPKM values should be restricted to specialty applications required by some users, _e.g._ manually comparing the expression levels of different genes or features. 

<div align="right">[Back to Table of Contents]()</div>

Read counting with _`summarizeOverlaps`_ using multiple nodes of a cluster
```{r read_counting_multinode, eval=FALSE}
library(BiocParallel)
f <- function(x) {
    library(systemPipeR); library(BiocParallel); library(GenomicFeatures)
    txdb <- loadDb("./data/tair10.sqlite")
    eByg <- exonsBy(txdb, by="gene")
    args <- systemArgs(sysma="tophat.param", mytargets="targets.txt")
    bfl <- BamFileList(outpaths(args), yieldSize=50000, index=character())
    summarizeOverlaps(eByg, bfl[x], mode="Union", ignore.strand=TRUE, inter.feature=TRUE, singleEnd=TRUE)
}
funs <- makeClusterFunctionsTorque("torque.tmpl")
param <- BatchJobsParam(length(args), resources=list(walltime="20:00:00", nodes="1:ppn=1", memory="6gb"), cluster.functions=funs)
register(param)
counteByg <- bplapply(seq(along=args), f) 
countDFeByg <- sapply(seq(along=counteByg), function(x) assays(counteByg[[x]])$counts)
rownames(countDFeByg) <- names(rowRanges(counteByg[[1]])); colnames(countDFeByg) <- names(outpaths(args))
```
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## Read counting for miRNA profiling experiments
Download miRNA genes from miRBase
```{r read_counting_mirna, eval=FALSE}
system("wget ftp://mirbase.org/pub/mirbase/19/genomes/My_species.gff3 -P ./data/")
gff <- import.gff("./data/My_species.gff3")
gff <- split(gff, elementMetadata(gff)$ID)
bams <- names(bampaths); names(bams) <- targets$SampleName
bfl <- BamFileList(bams, yieldSize=50000, index=character())
countDFmiR <- summarizeOverlaps(gff, bfl, mode="Union", ignore.strand=FALSE, inter.feature=FALSE) # Note: inter.feature=FALSE important since pre and mature miRNA ranges overlap
rpkmDFmiR <- apply(countDFmiR, 2, function(x) returnRPKM(counts=x, gffsub=gff))
write.table(assays(countDFmiR)$counts, "results/countDFmiR.xls", col.names=NA, quote=FALSE, sep="\t")
write.table(rpkmDFmiR, "results/rpkmDFmiR.xls", col.names=NA, quote=FALSE, sep="\t")
```
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## Correlation analysis of samples
The following computes the sample-wise Spearman correlation coefficients from the _`rlog`_ (regularized-logarithm) transformed expression values generated with the _`DESeq2`_ package. After transformation to a distance matrix, hierarchical clustering is performed with the _`hclust`_ function and the result is plotted as a dendrogram ([sample\_tree.pdf](./results/sample_tree.pdf)). 

```{r sample_tree_rlog, eval=TRUE}
library(DESeq2, warn.conflicts=FALSE, quietly=TRUE); library(ape, warn.conflicts=FALSE)
countDFpath <- system.file("extdata", "countDFeByg.xls", package="systemPipeR")
countDF <- as.matrix(read.table(countDFpath))
colData <- data.frame(row.names=targetsin(args)$SampleName, condition=targetsin(args)$Factor)
dds <- DESeqDataSetFromMatrix(countData = countDF, colData = colData, design = ~ condition)
d <- cor(assay(rlog(dds)), method="spearman")
hc <- hclust(dist(1-d))
plot.phylo(as.phylo(hc), type="p", edge.col=4, edge.width=3, show.node.label=TRUE, no.margin=TRUE)
```

<div align="center">**Figure 3:** Correlation dendrogram of samples for _`rlog`_ values. </div></br>

Alternatively, the clustering can be performed with _`RPKM`_ normalized expression values. In combination with Spearman correlation the results of the two clustering methods are often relatively similar. 

```{r sample_tree_rpkm, eval=FALSE}
rpkmDFeBygpath <- system.file("extdata", "rpkmDFeByg.xls", package="systemPipeR")
rpkmDFeByg <- read.table(rpkmDFeBygpath, check.names=FALSE)
rpkmDFeByg <- rpkmDFeByg[rowMeans(rpkmDFeByg) > 50,]
d <- cor(rpkmDFeByg, method="spearman")
hc <- hclust(as.dist(1-d))
plot.phylo(as.phylo(hc), type="p", edge.col="blue", edge.width=2, show.node.label=TRUE, no.margin=TRUE)
```

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## DEG analysis with _`edgeR`_
The following _`run_edgeR`_ function is a convenience wrapper for
identifying differentially expressed genes (DEGs) in batch mode with
_`edgeR`_'s GML method [@Robinson2010-uk] for any number of
pairwise sample comparisons specified under the _`cmp`_ argument. Users
are strongly encouraged to consult the 
[_`edgeR`_](\href{http://www.bioconductor.org/packages/devel/bioc/vignettes/edgeR/inst/doc/edgeRUsersGuide.pdf) vignette 
for more detailed information on this topic and how to properly run _`edgeR`_ 
on data sets with more complex experimental designs. 
```{r edger_wrapper, eval=TRUE}
targets <- read.delim(targetspath, comment="#")
cmp <- readComp(file=targetspath, format="matrix", delim="-")
cmp[[1]]
countDFeBygpath <- system.file("extdata", "countDFeByg.xls", package="systemPipeR")
countDFeByg <- read.delim(countDFeBygpath, row.names=1)
edgeDF <- run_edgeR(countDF=countDFeByg, targets=targets, cmp=cmp[[1]], independent=FALSE, mdsplot="")
```

Filter and plot DEG results for up and down regulated genes. Because of the small size of the toy data set used by this vignette, the _FDR_ value has been set to a relatively high threshold (here 10%). More commonly used _FDR_ cutoffs are 1% or 5%. The definition of '_up_' and '_down_' is given in the corresponding help file. To open it, type _`?filterDEGs`_ in the R console. 
```{r edger_deg_counts, eval=TRUE}
DEG_list <- filterDEGs(degDF=edgeDF, filter=c(Fold=2, FDR=10))
```
<div align="center">**Figure 4:** Up and down regulated DEGs identified by _`edgeR`_. </div></br>

```{r edger_deg_stats, eval=TRUE}
names(DEG_list)
DEG_list$Summary[1:4,]
```
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## DEG analysis with _`DESeq2`_ 
The following _`run_DESeq2`_ function is a convenience wrapper for
identifying DEGs in batch mode with _`DESeq2`_ [@Love2014-sh] for any number of
pairwise sample comparisons specified under the _`cmp`_ argument. Users
are strongly encouraged to consult the 
[_`DESeq2`_](http://www.bioconductor.org/packages/devel/bioc/vignettes/DESeq2/inst/doc/DESeq2.pdf) vignette
for more detailed information on this topic and how to properly run _`DESeq2`_ 
on data sets with more complex experimental designs. 
```{r deseq2_wrapper, eval=TRUE}
degseqDF <- run_DESeq2(countDF=countDFeByg, targets=targets, cmp=cmp[[1]], independent=FALSE)
```

Filter and plot DEG results for up and down regulated genes. 
```{r deseq2_deg_counts, eval=TRUE}
DEG_list2 <- filterDEGs(degDF=degseqDF, filter=c(Fold=2, FDR=10))
```
<div align="center">**Figure 5:** Up and down regulated DEGs identified by _`DESeq2`_. </div></br>

<div align="right">[Back to Table of Contents]()</div>

## Venn Diagrams
The function _`overLapper`_ can compute Venn intersects for large numbers of sample sets (up to 20 or more) and _`vennPlot`_ can plot 2-5 way Venn diagrams. A useful feature is the possiblity to combine the counts from several Venn comparisons with the same number of sample sets in a single Venn diagram (here for 4 up and down DEG sets).
```{r vennplot, eval=TRUE}
vennsetup <- overLapper(DEG_list$Up[6:9], type="vennsets")
vennsetdown <- overLapper(DEG_list$Down[6:9], type="vennsets")
vennPlot(list(vennsetup, vennsetdown), mymain="", mysub="", colmode=2, ccol=c("blue", "red"))
```
<div align="center">**Figure 6:** Venn Diagram for 4 Up and Down DEG Sets. </div></br>

<div align="right">[Back to Table of Contents]()</div>

## GO term enrichment analysis of DEGs
### Obtain gene-to-GO mappings
The following shows how to obtain gene-to-GO mappings from _`biomaRt`_ (here for _A. thaliana_) and how to organize them for the downstream GO term enrichment analysis. Alternatively, the gene-to-GO mappings can be obtained for many organisms from Bioconductor's  _`*.db`_ genome annotation packages or GO annotation files provided by various genome databases. For each annotation this relatively slow preprocessing step needs to be performed only once. Subsequently, the preprocessed data can be loaded with the _`load`_ function as shown in the next subsection. 
```{r get_go_biomart, eval=FALSE}
library("biomaRt")
listMarts() # To choose BioMart database
m <- useMart("ENSEMBL_MART_PLANT"); listDatasets(m) 
m <- useMart("ENSEMBL_MART_PLANT", dataset="athaliana_eg_gene")
listAttributes(m) # Choose data types you want to download
go <- getBM(attributes=c("go_accession", "tair_locus", "go_namespace_1003"), mart=m)
go <- go[go[,3]!="",]; go[,3] <- as.character(go[,3])
dir.create("./data/GO")
write.table(go, "data/GO/GOannotationsBiomart_mod.txt", quote=FALSE, row.names=FALSE, col.names=FALSE, sep="\t")
catdb <- makeCATdb(myfile="data/GO/GOannotationsBiomart_mod.txt", lib=NULL, org="", colno=c(1,2,3), idconv=NULL)
save(catdb, file="data/GO/catdb.RData") 
```
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### Batch GO term enrichment analysis
Apply the enrichment analysis to the DEG sets obtained in the above differential expression analysis. Note, in the following example the _FDR_ filter is set here to an unreasonably high value, simply because of the small size of the toy data set used in this vignette. Batch enrichment analysis of many gene sets is performed with the _`GOCluster_Report`_ function. When _`method="all"`_, it returns all GO terms passing the p-value cutoff specified under the _`cutoff`_ arguments. When _`method="slim"`_, it returns only the GO terms specified under the _`myslimv`_ argument. The given example shows how one can obtain such a GO slim vector from BioMart for a specific organism.  
```{r go_enrichment, eval=FALSE}
load("data/GO/catdb.RData")
DEG_list <- filterDEGs(degDF=edgeDF, filter=c(Fold=2, FDR=50), plot=FALSE)
up_down <- DEG_list$UporDown; names(up_down) <- paste(names(up_down), "_up_down", sep="")
up <- DEG_list$Up; names(up) <- paste(names(up), "_up", sep="")
down <- DEG_list$Down; names(down) <- paste(names(down), "_down", sep="")
DEGlist <- c(up_down, up, down)
DEGlist <- DEGlist[sapply(DEGlist, length) > 0]
BatchResult <- GOCluster_Report(catdb=catdb, setlist=DEGlist, method="all", id_type="gene", CLSZ=2, cutoff=0.9, gocats=c("MF", "BP", "CC"), recordSpecGO=NULL)
library("biomaRt"); m <- useMart("ENSEMBL_MART_PLANT", dataset="athaliana_eg_gene")
goslimvec <- as.character(getBM(attributes=c("goslim_goa_accession"), mart=m)[,1])
BatchResultslim <- GOCluster_Report(catdb=catdb, setlist=DEGlist, method="slim", id_type="gene", myslimv=goslimvec, CLSZ=10, cutoff=0.01, gocats=c("MF", "BP", "CC"), recordSpecGO=NULL)
```
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### Plot batch GO term results
The _`data.frame`_ generated by _`GOCluster_Report`_ can be plotted with the _`goBarplot`_ function. Because of the variable size of the sample sets, it may not always be desirable to show the results from different DEG sets in the same bar plot. Plotting single sample sets is achieved by subsetting the input data frame as shown in the first line of the following example. 
```{r plot_go_enrichment, eval=FALSE}
gos <- BatchResultslim[grep("M6-V6_up_down", BatchResultslim$CLID), ]
gos <- BatchResultslim
pdf("GOslimbarplotMF.pdf", height=8, width=10); goBarplot(gos, gocat="MF"); dev.off()
goBarplot(gos, gocat="BP")
goBarplot(gos, gocat="CC")
```
![](GOslimbarplotMF.png)
<div align="center">**Figure 7:** GO Slim Barplot for MF Ontology.</div></br>

<div align="right">[Back to Table of Contents]()</div>


## Clustering and heat maps
The following example performs hierarchical clustering on the _`rlog`_ transformed expression matrix subsetted by the DEGs identified in the 
above differential expression analysis. It uses a Pearson correlation-based distance measure and complete linkage for cluster joining.
```{r hierarchical_clustering, eval=FALSE}
library(pheatmap)
geneids <- unique(as.character(unlist(DEG_list[[1]])))
y <- assay(rlog(dds))[geneids, ]
pdf("heatmap1.pdf")
pheatmap(y, scale="row", clustering_distance_rows="correlation", clustering_distance_cols="correlation")
dev.off()
```
![](heatmap1.png)
<div align="center">**Figure 8:** Heat map with hierarchical clustering dendrograms of DEGs.</div></br>

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# Workflow templates

## RNA-Seq sample

Load the RNA-Seq sample workflow into your current working directory.
```{r genRna_workflow_single, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow="rnaseq")
setwd("rnaseq")
```
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### Run workflow
Next, run the chosen sample workflow _`systemPipeRNAseq`_ ([PDF](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/rnaseq/systemPipeRNAseq.pdf?raw=true), [Rnw](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/rnaseq/systemPipeRNAseq_single.Rnw)) by executing from the command-line _`make -B`_ within the _`rnaseq`_ directory. Alternatively, one can run the code from the provided _`*.Rnw`_ template file from within R interactively. 

Workflow includes following steps:

1. Read preprocessing
    + Quality filtering (trimming)
    + FASTQ quality report
2. Alignments: _`Tophat2`_ (or any other RNA-Seq aligner)
3. Alignment stats 
4. Read counting 
5. Sample-wise correlation analysis
6. Analysis of differentially expressed genes (DEGs)
7. GO term enrichment analysis
8. Gene-wise clustering

<div align="right">[Back to Table of Contents]()</div>

## ChIP-Seq sample

Load the ChIP-Seq sample workflow into your current working directory.
```{r genChip_workflow_single, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow="chipseq")
setwd("chipseq")
```
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### Run workflow
Next, run the chosen sample workflow _`systemPipeChIPseq_single`_ ([PDF](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/chipseq/systemPipeChIPseq.pdf?raw=true), [Rnw](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/chipseq/systemPipeChIPseq_single.Rnw)) by executing from the command-line _`make -B`_ within the _`chipseq`_ directory. Alternatively, one can run the code from the provided _`*.Rnw`_ template file from within R interactively. 

Workflow includes following steps:

1. Read preprocessing
    + Quality filtering (trimming)
    + FASTQ quality report
2. Alignments: _`Bowtie2`_ or _`rsubread`_
3. Alignment stats 
4. Peak calling: _`MACS2`_, _`BayesPeak`_ 
5. Peak annotation with genomic context
6. Differential binding analysis
7. GO term enrichment analysis
8. Motif analysis

<div align="right">[Back to Table of Contents]()</div>

## VAR-Seq sample 

### VAR-Seq workflow for single machine

Load the VAR-Seq sample workflow into your current working directory.
```{r genVar_workflow_single, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow="varseq")
setwd("varseq")
```
<div align="right">[Back to Table of Contents]()</div>

### Run workflow
Next, run the chosen sample workflow _`systemPipeVARseq_single`_ ([PDF](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/varseq/systemPipeVARseq_single.pdf?raw=true), [Rnw](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/varseq/systemPipeVARseq_single.Rnw)) by executing from the command-line _`make -B`_ within the _`varseq`_ directory. Alternatively, one can run the code from the provided _`*.Rnw`_ template file from within R interactively. 

Workflow includes following steps:

1. Read preprocessing
    + Quality filtering (trimming)
    + FASTQ quality report
2. Alignments: _`gsnap`_, _`bwa`_
3. Variant calling: _`VariantTools`_, _`GATK`_, _`BCFtools`_
4. Variant filtering: _`VariantTools`_ and _`VariantAnnotation`_
5. Variant annotation: _`VariantAnnotation`_
6. Combine results from many samples
7. Summary statistics of samples

<div align="right">[Back to Table of Contents]()</div>

### VAR-Seq workflow for computer cluster
The workflow template provided for this step is called _`systemPipeVARseq.Rnw`_ ([PDF](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/varseq/systemPipeVARseq.pdf?raw=true), [Rnw](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/varseq/systemPipeVARseq.Rnw)).
It runs the above VAR-Seq workflow in parallel on multiple computer nodes of an HPC system using Torque as scheduler. 

<div align="right">[Back to Table of Contents]()</div>

## Ribo-Seq sample

Load the Ribo-Seq sample workflow into your current working directory.
```{r genRibo_workflow_single, eval=FALSE}
library(systemPipeRdata)
genWorkenvir(workflow="riboseq")
setwd("riboseq")
```
<div align="right">[Back to Table of Contents]()</div>

### Run workflow
Next, run the chosen sample workflow _`systemPipeRIBOseq`_ ([PDF](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/riboseq/systemPipeRIBOseq.pdf?raw=true), [Rnw](https://github.com/tgirke/systemPipeRdata/blob/master/inst/extdata/workflows/ribseq/systemPipeRIBOseq_single.Rnw)) by executing from the command-line _`make -B`_ within the _`ribseq`_ directory. Alternatively, one can run the code from the provided _`*.Rnw`_ template file from within R interactively. 

Workflow includes following steps:

1. Read preprocessing
    + Adaptor trimming and quality filtering
    + FASTQ quality report
2. Alignments: _`Tophat2`_ (or any other RNA-Seq aligner)
3. Alignment stats
4. Compute read distribution across genomic features
5. Adding custom features to workflow (e.g. uORFs)
6. Genomic read coverage along transcripts
7. Read counting 
8. Sample-wise correlation analysis
9. Analysis of differentially expressed genes (DEGs)
10. GO term enrichment analysis
11. Gene-wise clustering
12. Differential ribosome binding (translational efficiency)

<div align="right">[Back to Table of Contents]()</div>


# Version information

```{r sessionInfo}
sessionInfo()
```
<div align="right">[Back to Table of Contents]()</div>

# References
