Note: if you use systemPipeR in published research, please cite: Backman, T.W.H and Girke, T. (2016). systemPipeR: NGS Workflow and Report Generation Environment. BMC Bioinformatics, 17: 388. 10.1186/s12859-016-1241-0.


systemPipeR provides flexible utilities for building and running automated end-to-end analysis workflows for a wide range of research applications, including next-generation sequencing (NGS) experiments, such as RNA-Seq, ChIP-Seq, VAR-Seq and Ribo-Seq (H Backman and Girke 2016). Important features include a uniform workflow interface across different data analysis 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 (Figure 1). 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 (Heng Li 2013; H. Li and Durbin 2009), HISAT2 (Kim, Langmead, and Salzberg 2015), TopHat2 (Kim et al. 2013) and Bowtie2 (Langmead and Salzberg 2012), as well as the R-based NGS aligners Rsubread* (Liao, Smyth, and Shi 2013) and gsnap (gmapR) (Wu and Nacu 2010). Efficient handling of complex sample sets (e.g. FASTQ/BAM files) and experimental designs are facilitated by a well-defined sample annotation infrastructure which improves reproducibility and user-friendliness of many typical analysis workflows in the NGS area (Lawrence et al. 2013).

The main motivation and advantages of using systemPipeR for complex data analysis tasks are:

  1. Facilitates the 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 the complexity of using compute clusters for R and command-line software
  6. Accelerates runtime of workflows via parallelization on computer systems with multiple CPU cores and/or multiple compute nodes
  7. Improves reproducibility by automating analyses and generation of analysis reports

Figure 1: Relevant features in systemPipeR. Workflow design concepts are illustrated under (A & B). Examples of systemPipeR’s visualization functionalities are given under (C).

A central concept for designing workflows within the systemPipeR environment is the use of workflow management containers. In previous versions, systemPipeR used a custom command-line interface called SYSargs (see Figure 3) and for this purpose will continue to be supported for some time. With the latest Bioconductor Release 3.9, we are adopting for this functionality the widely used community standard Common Workflow Language (CWL) for describing analysis workflows in a generic and reproducible manner, introducing SYSargs2 workflow control class (see Figure 2). Using this community standard in systemPipeR has many advantages. For instance, the integration of CWL allows running systemPipeR workflows from a single specification instance either entirely from within R, from various command-line wrappers (e.g., cwl-runner) or from other languages (, e.g., Bash or Python). systemPipeR includes support for both command-line and R/Bioconductor software as well as resources for containerization, parallel evaluations on computer clusters along with the automated generation of interactive analysis reports.

An important feature of systemPipeR's CWL interface is that it provides two options to run command-line tools and workflows based on CWL. First, one can run CWL in its native way via an R-based wrapper utility for cwl-runner or cwl-tools (CWL-based approach). Second, one can run workflows using CWL’s command-line and workflow instructions from within R (R-based approach). In the latter case the same CWL workflow definition files (e.g. *.cwl and *.yml) are used but rendered and executed entirely with R functions defined by systemPipeR, and thus use CWL mainly as a command-line and workflow definition format rather than software to run workflows. In this regard systemPipeR also provides several convenience functions that are useful for designing and debugging workflows, such as a command-line rendering function to retrieve the exact command-line strings for each data set and processing step prior to running a command-line.

This overview introduces the design of a new CWL S4 class in systemPipeR, as well as the custom command-line interface, combined with the overview of all the common analysis steps of NGS experiments.

Workflow design structure using SYSargs2

The flexibility of systemPipeR's new interface workflow control class is the driving factor behind the use of as many steps necessary for the analysis, as well as the connection between command-line- or R-based software. The connectivity among all workflow steps is achieved by the SYSargs2 workflow control class (see Figure 3). This S4 class is a list-like container where each instance stores all the input/output paths and parameter components required for a particular data analysis step. SYSargs2 instances are generated by two constructor functions, loadWorkflow and renderWF, using as data input targets or yaml files as well as two cwl parameter files (for details see below). When running preconfigured workflows, the only input the user needs to provide is the initial targets file containing the paths to the input files (e.g. FASTQ) along with unique sample labels. Subsequent targets instances are created automatically. The parameters required for running command-line software is provided by the parameter (.cwl) files described below.

We also introduce the SYSargs2Pipe class that organizes one or many SYSargs2 containers in a single compound object capturing all information required to run, control and monitor complex workflows from start to finish. This design enhances the systemPipeR workflow framework with a generalized, flexible, and robust design.

Figure 2: Workflow steps with input/output file operations are controlled by SYSargs2 objects. Each SYSargs2 instance is constructed from one targets and two param files. The only input provided by the user is the initial targets file. Subsequent targets instances are created automatically, from the previous output files. Any number of predefined or custom workflow steps are supported. One or many SYSargs2 objects are organized in an SYSargs2Pipe container.

Workflow Management using SYSargsList

systemPipeR allows creation (multi-step analyses) and execution of workflow entirely for R, with control, flexibility, and scalability of the all process. The execution of the workflow can be sent to a HPC, can be parallelizes, accelerating results acquisition. A workflow management system provides an infrastructure for the set-up, performance and monitoring of a defined sequence of tasks, arranged as a workflow application.

Figure 3: Workflow Management using SYSargsList.

Workflow design structure using SYSargs: Previous version

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/output 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 combination of command-line or R-based software.

Figure 4: Workflow design structure of systemPipeR using SYSargs.


H Backman, Tyler W, and Thomas Girke. 2016. “systemPipeR: NGS workflow and report generation environment.” BMC Bioinformatics 17 (1): 388.

Kim, Daehwan, Ben Langmead, and Steven L Salzberg. 2015. “HISAT: A Fast Spliced Aligner with Low Memory Requirements.” Nat. Methods 12 (4): 357–60.

Kim, Daehwan, Geo Pertea, Cole Trapnell, Harold Pimentel, Ryan Kelley, and Steven L Salzberg. 2013. “TopHat2: Accurate Alignment of Transcriptomes in the Presence of Insertions, Deletions and Gene Fusions.” Genome Biol. 14 (4): R36.

Langmead, Ben, and Steven L Salzberg. 2012. “Fast Gapped-Read Alignment with Bowtie 2.” Nat. Methods 9 (4): 357–59.

Lawrence, Michael, Wolfgang Huber, Hervé Pagès, Patrick Aboyoun, Marc Carlson, Robert Gentleman, Martin T Morgan, and Vincent J Carey. 2013. “Software for Computing and Annotating Genomic Ranges.” PLoS Comput. Biol. 9 (8): e1003118.

Li, H, and R Durbin. 2009. “Fast and Accurate Short Read Alignment with Burrows-Wheeler Transform.” Bioinformatics 25 (14): 1754–60.

Li, Heng. 2013. “Aligning Sequence Reads, Clone Sequences and Assembly Contigs with BWA-MEM.” arXiv [q-Bio.GN], March.

Liao, Yang, Gordon K Smyth, and Wei Shi. 2013. “The Subread Aligner: Fast, Accurate and Scalable Read Mapping by Seed-and-Vote.” Nucleic Acids Res. 41 (10): e108.

Wu, T D, and S Nacu. 2010. “Fast and SNP-tolerant Detection of Complex Variants and Splicing in Short Reads.” Bioinformatics 26 (7): 873–81.