VANIR - NextFlow pipeline for Viral Variant calling and de novo Assembly of Nanopore and Illumina Reads for high-quality genomes
If any issue raises, please do not hesitate to open an issue in this repository or to contact me at joan.marti.carreras@gmail.com
The aim of this pipeline is to accuratley base-call and QC read information and de novo assemble long-read data from Oxford Nanopore Technologies (ONT) reads to yield a complete dsDNA viral genome. The target genome is later corrected using multiple algorithms (Racon and Medaka) and a final polishing using a higher accuracy read data set from Illumina.
Later single nucleotide variants (SNV) and structural variants (SV) are called from Illumina and Nanopore reads, respectivley using a user-provided reference and to assess differences against standard and using the de novo assembled genome in order to assess the diversity within the sample.
The pipeline is writen in NextFlow pipeline manager. This framework allow users to easly launch and coordinate the pipeline in different environments. It can easly resume failed runs and provide basic metrics of the consumed resources (see options section).
Additionally, all software has been bundled in a Singularity container, containing its own operating system (Ubuntu 16.04) and all libraries and programs needed to run the pipeline. Singularity makes this pipeline highly transportable, as can be run it creates its own environemnt; convenient, as everything has been pre-bundled for direct user usage; and reproducible, as libraries and programs are independent from general installation and will run always on the same manner.
This pipeline is partially GPU-enabled, concerning maninly ONT basecalling and ONT read correction with Medaka.
This pipeline is a working in progress on v1.0.
Only the Singulairy image needs to be installed. For this first install Singularity as follows:
Open the terminal and type the following:
VERSION=3.5.2
wget https://github.com/singularityware/singularity/releases/download/$VERSION/singularity-$VERSION.tar.gz
tar xvf singularity-$VERSION.tar.gz
cd singularity-$VERSION
./configure --prefix=/usr/local
make
sudo make install
Open the terminal and type the following:
/usr/bin/ruby -e "$(curl -fsSL https://raw.githubusercontent.com/Homebrew/install/master/install)"
brew cask install virtualbox
brew cask install vagrant
brew cask install vagrant-manager
Download Singularity from here
mkdir singularity-vm
cd singularity-vm
vagrant destroy
vagrant init singularityware/singularity-3.5.2
vagrant up
vagrant ssh
First install Git, VirtualBox, Vagrant and Vagrant Manager.
Then install Singularity, and run on GitBash:
mkdir singularity-vm
cd singularity-vm
vagrant destroy
vagrant init singularityware/singularity-3.5.2
vagrant up
vagrant ssh
After installing Singularity, VANIR image must be build from recepie or downloaded.
To build from recepie please follow:
git clone https://github.com/joanmarticarreras/VANIR.git
cd VANIR/
sudo singularity build --writable VANIR singularity-recepie/VANIR-singularity-recepie.txt
sudo cp main.nf VANIR/nextflow/
There are 2 sets of options, one comming from VANIR pipeline itself and one from the NextFlow manager:
Folder to raw fast5 reads [PATH]: --fast5
Illumina 1st pair-end reads (can be .gz) [FILE]: --read1
Illumina 2nd pair-end reads (can be .gz) [FILE]: --read2
Illumina adapters [FILE]: --illumina_adapters
Sample prefix [FILE]: --prefix
Reference genome [FILE]: --reference
Sequencing contaminants [FILE]: --contaminants
Number of CPUs asked [NUMERIC]: --cpu
GPU-enabled ('true' enabled // 'false' disabled) [Boolean]: --gpu
Guppy-basecalling mode ('precise' for hac // 'fast' for fast) [Boolean]: --guppy_algorithm
Expected genome size (STRING, eg 35k, 8g or 250m): --genome_size
Produce an html report with useful metrics of the pipeline [FILE] -with-report
Produce a tabular file with tracings of each processes [FILE] -with-trace
Produce an html graphic of all process executed [FILE] -with-timeline
Produce a graph-image (.dot/.html/.pdf/.png/.svg) of the pipeline [FILE] -with-dag
To run this pipeline, run on terminal:
sudo singularity shell -B <BIND-PATH-DATA> --nv --writable VANIR
nextflow run nextflow/main.nf OPTIONS
Option -B or bind, can be used as many times needed, specifying different paths for data to be mounted in specific locations in the Singularity container. Option --nv is mandatory for GPU computing, and --writable options guarantees at this first release of the pipeline to write within the image. Later results can be exported.
Results and information regarding the publication of VANIR can be found in the manuscript-data/ folder.