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Full article title systemPipeR: NGS workflow and report generation environment
Journal BMC Bioinformatics
Author(s) Backman, Tyler W.H.; Girke, Thomas
Author affiliation(s) University of California, Riverside
Primary contact Email: thomas dot girke at ucr dot edu
Year published 2016
Volume and issue 17
Page(s) 388
DOI 10.1186/s12859-016-1241-0
ISSN 1471-2105
Distribution license Creative Commons Attribution 4.0 International
Website https://bmcbioinformatics.biomedcentral.com/articles/10.1186/s12859-016-1241-0
Download https://bmcbioinformatics.biomedcentral.com/track/pdf/10.1186/s12859-016-1241-0 (PDF)
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Abstract

Background: Next-generation sequencing (NGS) has revolutionized how research is carried out in many areas of biology and medicine. However, the analysis of NGS data remains a major obstacle to the efficient utilization of the technology, as it requires complex multi-step processing of big data, demanding considerable computational expertise from users. While substantial effort has been invested on the development of software dedicated to the individual analysis steps of NGS experiments, insufficient resources are currently available for integrating the individual software components within the widely used R/Bioconductor environment into automated workflows capable of running the analysis of most types of NGS applications from start-to-finish in a time-efficient and reproducible manner.

Results: To address this need, we have developed the R/Bioconductor package systemPipeR. It is an extensible environment for both building and running end-to-end analysis workflows with automated report generation for a wide range of NGS applications. Its unique features include a uniform workflow interface across different NGS applications, automated report generation, and support for running both R and command-line software on local computers and computer clusters. A flexible sample annotation infrastructure efficiently handles complex sample sets and experimental designs. To simplify the analysis of widely used NGS applications, the package provides pre-configured workflows and reporting templates for RNA-Seq, ChIP-Seq, VAR-Seq, and Ribo-Seq. Additional workflow templates will be provided in the future.

Conclusions: systemPipeR accelerates the extraction of reproducible analysis results from NGS experiments. By combining the capabilities of many R/Bioconductor and command-line tools, it makes efficient use of existing software resources without limiting the user to a set of predefined methods or environments. systemPipeR is freely available for all common operating systems from Bioconductor (http://bioconductor.org/packages/devel/systemPipeR).

Keywords: analysis workflow, next generation sequencing (NGS), Ribo-Seq, ChIP-Seq, RNA-Seq, VAR-Seq

Background

By allowing scientists to rapidly sequence and quantify DNA and RNA molecules, next-generation sequencing (NGS) technology has transformed biology into one of the most data intensive research disciplines. In the past, experiments have been performed on a gene-by-gene basis, while NGS has introduced an age where it is has become a routine to sequence entire transcriptomes, genomes, or epigenomes rather than their isolated parts of interest. It will soon be possible to conduct these experiments on large numbers of single cell samples[1][2] for a wide range of time points, treatments, and genetic backgrounds to study biological systems with greater resolution and precision. Sequencing the genetic material of each individual within entire populations of organisms of the same species or genus will enable the study of adaptation processes[3], disease progression, and micro-evolution in real time.[4] This technological shift empowers researchers to address questions at a genome-wide scale, for example by profiling the mRNA, miRNA, and DNA methylation states of a large set of biological samples in parallel.[5]

The success of NGS-driven research has led to a data explosion of increasing size and complexity, making it now more time-consuming and challenging for researchers to extract knowledge from their experiments. Rapid processing of the results is essential to test, refine, and formulate new hypotheses for designing follow-up experiments. As a result, biologists have to dedicate nowadays substantial time to data analysis tasks while training themselves effectively as genome data scientists rather than focusing on experimentation as they used to in the past.

In recent years, a considerable number of algorithms, statistical methods, and software tools has been developed to perform the individual analysis steps of different NGS applications. These include short read pre-processors, aligners, variant and peak callers, as well as statistical methods for the analysis of genomic regions that are differentially expressed[6][7], bound[8], or methylated.[9][10] Also essential are tools for processing short read alignments[11], genomic intervals[12][13], and annotations.[14] However, most data analysis routines of NGS applications are very complex, involving multiple software tools for their many processing steps. As a result, there is a great need for flexible software environments connecting the individual software components to automated workflows in order to perform complex genome-wide analyses in an efficient and reproducible manner. While many workflow management resources exist[15][16][17][18][19][20][21][22][23][24] for a variety of data analysis programming languages (for details see below), only insufficient general purpose NGS workflow solutions are currently available for the popular R programming language. R and the affiliated Bioconductor environment provide a substantial number of widely used tools with a large user base in this area.[10] Thus, a workflow framework for federating NGS applications from within R will have many benefits for experimental and computational scientists who use R for NGS data analysis.

To address this need, we designed systemPipeR as a Bioconductor package for building and running workflows for most NGS applications, with support for integrating a wide array of command-line and R/Bioconductor software.

Implementation

Environment

systemPipeR has been implemented as an open-source Bioconductor package using the R programming language for statistical computing and graphics. R was chosen as the core development platform for systemPipeR because of the following reasons. (i) R is currently one of the most popular statistical data analysis and programming environments in bioinformatics. (ii) Its external language bindings support the implementation of computationally time-consuming analysis steps in high-performance languages such as C/C++. (iii) It supports advanced parallel computation on multi-core machines and computer clusters. (iv) A well developed infrastructure interfaces R with several other popular programing languages such as Python. (v) R provides advanced graphical and visualization utilities for scientific computing. (vi) It offers access to a vast landscape of statistical and machine learning tools. (vii) Its integration with the Bioconductor project promotes reusability of genomics software components, while also making efficient use of a large number of existing NGS packages that are well tested and widely used by the community. To support long-term reproducibility of analysis outcomes, systemPipeR is also distributed as a Docker image of Bioconductor’s sequencing division. Docker containers provide an efficient solution for packaging complex software together with all its system dependencies to ensure it will run the same in the future across different environments, including different operating systems and cloud-based solutions.

Workflow design

systemPipeR workflows (Fig. 1) can be run from start-to-finish with a single command, or stepwise in interactive mode from the R console. New workflows are constructed, or existing ones modified, by connecting so-called SYSargs workflow control modules (R S4 class). Each SYSargs module contains instructions needed for processing a set of input files with a specific command-line or R software; as well as the paths to the corresponding outputs generated by a specific NGS tool such as a read preprocessor (trimmed/filtered FASTQ files), aligner (SAM/BAM files), read counter, variant caller (VCF/BCF files), peak caller (BED/WIG files), or statistical function. Typically, the only input the user needs to provide for running workflows is a single tabular targets file containing the paths to the initial sample input files (e.g. FASTQ) along with sample labels, and if appropriate biological replicate and contrast information for controlling differential abundance analyses (e.g., gene expression). Downstream derivatives of these targets files along with the corresponding SYSargs instances (see Fig. 1) are created automatically within each workflow.


Fig1 Backman BMCBio2016 17.gif

Figure 1. Workflow steps with input/output file operations are controlled by SYSargs objects. Each SYSargs instance is constructed from a targets and a param file. The only input required from the user is the initial targets file. Subsequent instances are created automatically. Any number of predefined or custom workflow steps is supported.

The parameters required for running command-line software are provided by parameter (param) files, described below. For R-based workflow steps, param files are not required but can be useful for operations importing and/or exporting sample-level files. This modular design has several advantages. First, it provides a high level of flexibility for designing workflows, such as allowing the user to start workflows from the very beginning or anywhere in-between (e.g. FASTQ or BAM level). Second, it is straightforward to add custom workflow steps without requiring computational expert knowledge from users. Workflows can also have any number of steps including branch points. Lastly, it also minimizes errors as all input and output files are registered, and sample labels specified in the initial targets file will be consistently used throughout all workflow results, including plots, tables, and workflow reports.

Command-line software support

An important feature of systemPipeR is support for running command-line software directly from R on both single machines or computer clusters. This offers several advantages, such as seamless integration of most command-line software available in the NGS field with the extensive genome analysis resources provided by R/Bioconductor. The user interface for running command-line software has been generalized as a single function for ease of use, while only one additional command will run the same tool in parallel mode on a computer cluster (see below). Examples of command-line software used by systemPipeR’s preconfigured workflow templates (see below) include the aligners BWA-MEM[25], Bowtie2[26], TopHat2[27], HISAT2[28], as well as the peak/variant callers MACS[29], GATK[30], and BCFtools.[11] Support for additional command-line NGS software can be added by simply providing the argument settings of a chosen software in a tabular param file. If appropriate, new param files can be permanently included in the package to share them with the community. Functionality for creating param files automatically will be provided in the future. This will allow users to create new param instances simply by providing an example of the command-line syntax of a chosen software tool.

Major advantages of running command-line software from within systemPipeR include: a uniform sample management infrastructure within and across workflows; integration of BatchJobs’[31] efficient error management infrastructure for job submissions on computer clusters; the simplicity of restarting failed processes; as well as seamless addition of new samples (e.g., FASTQ or BAM files). In case of a restart, the system will skip the analysis steps of already completed samples and only perform the analysis of the missing ones. If required, any workflow step can be rerun on demand for all or a subset of samples. When submitting command-line software to computer clusters, BatchJobs monitors the status of job submissions and alerts users of exceptions, while recording warning and error messages for each process in a log directory with a database-like structure that is accessible from within R or the command-line. This organization helps to diagnose and resolve errors.

Parallel evaluation

The processing time for NGS experiments can be greatly reduced by making use of parallel evaluation across several CPU cores on single machines, or multiple nodes of computer clusters and cloud-based systems. systemPipeR simplifies these parallelization tasks without creating any limitations for users who do not have access to high-performance computer (HPC) resources by providing the option to run workflows in serial or parallel mode. The parallelization functionalities available in systemPipeR are largely based on existing and well maintained R packages, mainly BatchJobs and BiocParallel.[31] By making use of cluster template files, most schedulers and queuing systems are also supported (e.g., Torque, Sun Grid Engine, Slurm). If required, entire workflows can be executed in parallel mode by issuing a single command, while simultaneously generating a detailed analysis report (for details see below). If sufficient parallel computer resources are available, systemPipeR can complete the entire analysis workflow of several complex NGS experiments, each containing large numbers of FASTQ files, within hours rather than days or weeks, as can be the case for non-parallelized workflows.

Automated analysis reports

systemPipeR generates automated analysis reports with knitr and R markdown.[32] These modern reporting environments integrate R code with LaTeX or Markdown. During the evaluation of the R code, reports are dynamically generated in PDF or HTML format. A caching system allows to re-execute selected workflow reporting steps without repeating unnecessary components. This way one can generate reports that resemble a research paper where user generated text is combined with analysis results. This includes support for citations, autogenerated bibliographies, code chunks with syntax highlighting, and inline evaluation of variables to update text content. Data components in a report such as tables and figures are updated automatically when rebuilding the document and/or rerunning workflows partially or entirely.

Results and discussion

Overview

systemPipeR provides utilities for building and running NGS analysis workflows. To adapt to community standards, widely used R/Bioconductor packages are integrated where possible. This includes the Bioconductor packages ShortRead, Biostrings, and Rsamtools for processing sequence and alignment files[33]; GenomicRanges, GenomicAlignments, and GenomicFeatures for handling genomic range operations, read counting, and annotation data[12]; edgeR and DESeq2 for differential abundance analysis[6][7]; and VariantTools and VariantAnnotation for filtering and annotating genome variants.[34] If necessary, one can substitute most of these packages with alternative R or command-line tools.

Because many NGS applications share overlapping analysis needs (Fig. 2 a), certain workflow steps are conceptualized in systemPipeR by a single generic function, with support for application-specific parameter settings (Table 1). For instance, most NGS applications involve a short read alignment step (see Fig. 2 b), but with very distinct mapping requirements, such as splice junction awareness and variant tolerance for RNA-Seq and VAR-Seq, respectively. To simplify their execution for the user, the different aligners can be run with the same runCommandline function where the software, and its parameter settings are specified in the corresponding SYSargs instance (see above and Fig. 1).


Fig2 Backman BMCBio2016 17.gif

Figure 2. Workflow Steps and Graphical Features. Relevant workflow steps of several NGS applications (a) are illustrated in form of a simplified flowchart (b). Examples of systemPipeR’s functionalities are given under (c) including: (1) eight different plots for summarizing the quality and diversity of short reads provided as FASTQ files; (2) strand-specific read count summaries for all feature types provided by a genome annotation; (3) summary plots of read depth coverage for any number of transcripts with nucleotide resolution upstream/downstream of their start and stop codons, as well as binned coverage for their coding regions; (4) enumeration of up- and down-regulated DEGs for user defined sample comparisons; (5) similarity clustering of sample profiles; (6) 2-5-way Venn diagrams for DEGs, peak and variant sets; (7) gene-wise clustering with a wide range of algorithms; and (8) support for plotting read pileups and variants in the context of genome annotations along with genome browser support.

Table 1. Selected functions. The table lists a subset of over 50 methods and functions defined by systemPipeR. Usage instructions are provided in the corresponding help pages and vignettes of the package.
Function name Description
genWorkenvir Generates workflow templates provided by systemPipeRdata helper package
systemArgs Constructs SYSargs workflow control module (S4 object) from targets and param files
runCommandline/tt> Executes command-line software on samples and parameters specified in SYSargs
clusterRun/tt> Runs command-line software in parallel mode on a computer cluster
clusterRun/tt> Runs command-line software in parallel mode on a computer cluster
preprocessReads/tt> Filtering and/or trimming of short reads using predefined or custom parameters
seeFASTQ/seeFASTQplot/tt> Generates quality reports for any number of FASTQ files
alignStats/tt> Generates alignment statistics, such as total number of reads and alignment frequency
run_edgeR/run_DESeq2/tt> Runs edgeR or DESeq2 for any number of pairwise sample comparisons
filterDEGs/tt> Filters and plots DEG results based on user-defined parameters
overLapper/vennPlot/tt> Computation of Venn intersects for 2-20 or more samples and 2-5 way Venn diagrams
GOCluster_Report/tt> GO term enrichment analysis for large numbers of gene sets
variantReport/tt> Generates a variant report containing genomic annotations and confidence statistics
predORF Prediction of short open reading frames in DNA sequences
featuretypeCounts Computes and plots read distribution for many feature types at once
featureCoverage Computes and plots read depth coverage from many transcripts

References

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Notes

This presentation is faithful to the original, with only a few minor changes to presentation. In some cases important information was missing from the references, and that information was added.

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