This fork of the iVar software simplifies the variant calling pipeline Snakemake so that replicate samples are no required, and the default naming conversion for Illumin a MiSeq FASTQ files is honoured. You should be able to run this on the output of a bcl2fastq conversion.

iVar is a computational package that contains functions broadly useful for viral amplicon-based sequencing. Additional tools for metagenomic sequencing are actively being incorporated into iVar. While each of these functions can be accomplished using existing tools, iVar contains an intersection of functionality from multiple tools that are required to call iSNVs and consensus sequences from viral sequencing data across multiple replicates. We implemented the following functions in iVar: (1) trimming of primers and low-quality bases, (2) consensus calling, (3) variant calling - both iSNVs and insertions/deletions, and (4) identifying mismatches to primer sequences and excluding the corresponding reads from alignment files.

An amplicon-based sequencing framework for accurately measuring intrahost virus diversity using PrimalSeq and iVar

Genome Biology 2019 20:8

Nathan D Grubaugh, Karthik Gangavarapu, Joshua Quick, Nathaniel L Matteson, Jaqueline Goes De Jesus, Bradley J Main, Amanda L Tan, Lauren M Paul, Doug E Brackney, Saran Grewal, Nikos Gurfield, Koen KA Van Rompay, Sharon Isern, Scott F Michael, Lark L Coffey, Nicholas J Loman, Kristian G Andersen

bioRxiv doi: https://doi.org/10.1101/383513

Manual for iVar is available here.

• HTSlib
• GCC any version after v5.0. Support for C++11 standard required.

Note:

• It is highly recommended that samtools also be installed alongside iVar. iVar uses the output of samtools mpileup to call variants and generate consensus sequences. In addition, samtools sort and index commands are very useful to setup a pipeline using iVar.

iVar is available on bioconda. To install conda, please use the miniconda package. After intalling conda please add the following channels,

conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge

To install iVar,

conda install ivar

iVar can be installed using Homebrew.

brew install brewsci/bio/ivar

Xcode from Apple is required to compile iVar (and other tools) from source. If you don't want to install the full Xcode package from the AppStore, you can install the Xcode command line tools,

xcode-select --install

GNU Autotools is required to compile iVar from source.

To install Autotools using homebrew please use the command below,

brew install autoconf automake libtool

HTSlib can be installed with conda using the command,

conda install -c bioconda htslib

The conda binary is by default installed at /opt/. You can check the installation location by running the following command,

which conda

The output of the command will be in this format - /opt/conda/bin/conda or /opt/anaconda2/bin/conda or /opt/anaconda3/bin/conda depending on whether you installed miniconda or anaconda.

If the output is for example, /opt/conda/bin/conda, then you can add the path to the lib folder to $LD_LIBRARY_PATH using the command below. You can add this to your ~/.bash_profile or ~/.bashrc to avoid rerunning the command everytime a new bash session starts.

export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:/opt/conda/lib

Installation instructions and downloads for HTSlib can be found at http://www.htslib.org/download/.

If HTSlib is installed in a non standard location, please add the following to your .bash_profile so that iVar can find HTSlib dynamic libraries during runtime.

export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:/path/to/hts/lib/folder

To install iVar, run the following commands.

./autogen.sh
./configure
make
make install

If HTSlib was installed using conda, please run the following commands by supplying the prefix to the bin folder of the conda binary.

The prefix to the bin folder can be found using the command which conda. The output of the command will be in this format - /opt/conda/bin/conda or /opt/anaconda2/bin/conda or /opt/anaconda3/bin/conda depending on whether you installed miniconda or anaconda. For example, if the output of the command is /opt/conda/bin/conda, the prefix to the htslib bin folder will be /opt/conda. This can be supplied to ./configure --with-hts=/opt/conda.

./autogen.sh
./configure --with-hts=/prefix/to/bin/folder/with/HTSlib
make
make install

If HTSlib was installed in a non standard location, please run the following commands,

./autogen.sh
./configure --with-hts=/prefix/to/bin/folder/with/HTSlib
make
make install

To test installation just run, ivar version and you should get the following output,

iVar version 1.0

Please raise issues and bug reports at https://github.com/andersen-lab/ivar/

GNU Autotools is required to compile iVar from source.

To install Autotools using APT please use the command below,

apt-get install autotools-dev

HTSlib can be installed with conda using the command,

conda install -c bioconda htslib

The conda binary is by default installed at /opt/. You can check the installation location by running the following command,

which conda

The output of the command will be in this format - /opt/conda/bin/conda or /opt/anaconda2/bin/conda or /opt/anaconda3/bin/conda depending on whether you installed miniconda or anaconda.

If the output is for example, /opt/conda/bin/conda, then you can add the path to the lib folder to $LD_LIBRARY_PATH using the command below. You can add this to your ~/.bash_profile or ~/.bashrc to avoid rerunning the command everytime a new bash session starts.

export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:/opt/conda/lib

Installation instructions and downloads for HTSlib can be found at http://www.htslib.org/download/.

If HTSlib is installed in a non standard location, please add the following to your .bash_profile so that iVar can find HTSlib dynamic libraries during runtime.

export LD_LIBRARY_PATH=$LD_LIBRARY_PATH:/path/to/hts/lib/folder

To install iVar, run the following commands.

./autogen.sh
./configure
make
make install

If HTSlib was installed using conda, please run the following commands by supplying the prefix to the bin folder of the conda binary.

The prefix to the bin folder can be found using the command which conda. The output of the command will be in this format - /opt/conda/bin/conda or /opt/anaconda2/bin/conda or /opt/anaconda3/bin/conda depending on whether you installed miniconda or anaconda. For example, if the output of the command is /opt/conda/bin/conda, the prefix to the htslib bin folder will be /opt/conda. This can be supplied to ./configure --with-hts=/opt/conda.

./autogen.sh
./configure --with-hts=/prefix/to/bin/folder/with/HTSlib
make
make install

If HTSlib was installed in a non standard location, please run the following commands,

./autogen.sh
./configure --with-hts=/prefix/to/bin/folder/with/HTSlib
make
make install

To test installation just run, ivar version and you should get the following output,

iVar version 1.0

Please raise issues and bug reports at https://github.com/andersen-lab/ivar/

iVar can also be run via Docker. Pull the docker image from Docker Hub using the following command,

docker pull andersenlabapps/ivar

This docker image contains all the required dependencies to run iVar and the [pipelines](@ref cookbookpage) developed using iVar. You will have to attach a docker volume to get data into the docker container. Instructions to do so are in the Docker docs.

iVar on Docker Hub

For bug reports please email gkarthik[at]scripps.edu or raise an issue on Github.

This work was supported in part by NIH grants U19AI135995, R21AI137690, and UL1TR002550.