RNA-seq analysis pipelines

Small RNA pipelines have beed developed within Virtual Laboratory for Plant Breeding projects. The pipelines make use of mostly R and Python. The diagram that has been used to build these pipelines:

Currently the Basic QC, Mapping, Spike removal & visualization, normalization and pre- and post visualization are ready to use.

Every pipeline is run in separate environment. By encapsulating most of the dependecies together with analysis code.

Different levels of dependencies:

system level packages

python

• Python version
• Python moudule versions

both can be fixed with use of python virtual environment

R

• R version
• R packages version the system level R version (first one in the path) is used

Right versions of R packages are ensured with use of Packrat. That alows for building whole R environment with rigth R packages.
Packrat environment works at the level of directory.

# example basicQC analysis directory 
# with all necessary Packrat related files and directories 
.
├── basicQC.htm               # html file with QC report 
├── basicQC.md                # markdown file genereated by knitr 
├── basicQC.Rmd               # Rmarkdown defining the QC report 
├── figure                    # plots 
│   ├── A.design.plot-1.png
│   ├── C3plot-1.png
│   ├── D1plot-1.png
│   └── D2plot-1.png
├── get_experiment_fq.py      # script that imports read data form sff files into fq files 
├── packrat                   # dir with R environment related files 
│   ├── init.R                # script initializing Packrat environment 
│   ├── lib                   # 
│   ├── lib-ext               # R library 
│   ├── lib-R                 # 
│   ├── packrat.lock          # log of all the package versions used by this environment 
│   ├── packrat.opts          # packrat options 
│   └── src
├── raw                       # fastq files generated from sff files 
│   ├── N406T.fastq
│   ├── N540T.fastq
│   ├── N583T.fastq
│   └── sknas.fastq
├── README.md                 # project readme file 
├── seqdesign.txt             # experiment design file 
└── Snakefile                 # pipeline defined for Snakemake 

Pipelines and tools developed at the University of Amsterdam:

• faradr

  R package used across the pipelines.

• basicQC

  Quality control of raw reads in context of the experiment.

• rnaxcount

  Alignment and counting fo small RNA species.

• rnaxqc

  Quality control of small RNA counts in the context of the experiment.

• sRNA-norm

  Normalisation for counts based on spike-in counts.

Glossary:

Packrat

Packrat is a dependency management system for R. Read more.

Snakemake

Python module and library for building workflows. Project's aim is to reduce the complexity of creating workflows by providing a fast and comfortable execution environment, together with a clean and modern domain specific specification language (DSL) in python style. Read more.

virtualenv

A tool to create isolated Python environments. See Read more.