• Introduction
  • Installation
  • Testing
  • Usage
  • Method details
  • Output
  • Pipeline summary
  • Citations

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Introduction

rvfvtyping is a bioinformatics analysis pipeline for classification and phylogenetic lineage assignment of Rift Valley fever virus consensus genomes using the glycoprotein Gn/G2 gene found within the M-segment of the virus genome.

Classifying query sequences involves two steps. The first step is the identification of the virus species and the second is the assignment of Rift Valley fever virus lineages through phylogenetic analysis. Classification of query sequences is performed using diamond while phylogenetic assignment uses iqtree, and is largely adopted from the initial pangolin method developed by ine O'Toole.

The pipeline is built using Nextflow, a workflow tool to run tasks across multiple compute infrastructures in a very portable manner. It comes with docker containers making installation trivial and results highly reproducible.

A web application of the pipeline is hosted on a dedicated server at the University of KwaZulu Natal and can be found here

Installation

rvfvtyping runs on UNIX/LINUX systems. You will install Miniconda3 from here. Once Miniconda3 has been installed, proceed with pipeline installation

git clone https://github.com/ajodeh-juma/rvfvtyping.git cd rvfvtyping conda env create -n rvfvtyping-env -f environment.yml conda activate rvfvtyping-env

Testing

• Optional: Test the installation on a single FASTA

  nextflow run main.nf -profile test

• Optional: Test the installation on several FASTA sequence files

  nextflow run main.nf -profile test_full

Usage

For minimal pipeline options, use the --help flag e.g.

nextflow run main.nf --help

To see all the options, use the --show_hidden_params flag e.g.

nextflow run main.nf --help --show_hidden_params

A typical command to classify and assign lineages using the glycoprotein (Gn) classifier nextflow run main.nf \ --input 'data/test/*.fa' \ --segment Gn \ --outdir output-dir \ -work-dir work-dir \

Method details

The pipeline offers several parameters including as highlighted:

``` Input/output options --input [string] Input Fasta file for typing --segment [string] genomic segment of the virus. options are 'Gn', 'S', 'M' and 'L' --outdir [string] The output directory where the results will be saved. [default: ./results] --email [string] Email address for completion summary.

Diamond options --skip_diamond [boolean] Skip all DIAMOND BLAST against the pre-configured database.

```

mandatory parameters

| parameter | description | type | |-------------|:-------------------------------------------------------:|:---------:| | --input | Input Fasta file(s) format .fa or .fasta for typing | string | | --segment | genomic segment of the virus. Gn, S, M, L | string |

Output

Several output files will be generated including a comma-separated values file (lineages.csv) will be a csv file with taxon name and lineage assigned for each input query sequence per line

e.g.

| Query | Lineage | aLRT | UFbootstrap | Length | Ns(%) | Note | Yearfirst | Yearlast | Countries | | ----------- |:---------:|:----------:|:----------:|:----------:|:----------:|:----------:|:----------:|:----------:|:----------: | | DQ380218|G|84|70|3885|0.00|assigned (bootstrap value >= 70)|1969|1993|Senegal;CAR;Zimbabwe;Guinea| | HM587118|L|99|100|490|0.00|assigned (bootstrap value >= 70)|1963|1995|Zimbabwe;Egypt;South Africa;Kenya| | DQ380221|D|92|98|3885|0.00|assigned (bootstrap value >= 70)|1973|1973|CAR| | DQ380222|J|77|27|3885|0.00|unassigned (bootstrap value < 70)|||| | HM587045|B|89|97|490|0.00|assigned (bootstrap value >= 70)|1972|1972|Kenya| | DQ380189|L|99|100|3885|0.00|assigned (bootstrap value >= 70)|1963|1995|Zimbabwe;Egypt;South Africa;Kenya| | HM587125|O|92|98|490|0.00|assigned (bootstrap value >= 70)|1951|1951|South Africa| | HM587108|I|87|90|490|0.00|assigned (bootstrap value >= 70)|1955|1956|South Africa| | MG972973|C|88|96|3852|0.00|assigned (bootstrap value >= 70)|1976|2016|South Africa;Somalia;Uganda;Angola;Madagascar;Sudan;Zimbabwe;Mauritania;Saudi Arabia;Kenya| | AF134496|N|88|84|738|0.00|assigned (bootstrap value >= 70)|1975|1993|Senegal;Mauritania;Burkina Faso| | EU574086.1|J|74|33|1690|0.00|unassigned (bootstrap value < 70)|||| | RVFVNamibia2011MT561463NAM_2011|C|89|95|3830|0.00|assigned (bootstrap value >= 70)|1976|2016|South Africa;Somalia;Uganda;Angola;Madagascar;Sudan;Zimbabwe;Mauritania;Saudi Arabia;Kenya|

If --skip_diamond is not used, the classification file diamond_results.csv is not generated

| QueryID | Length | SubjectID | Segment | Product | PercentIdentity | Mismatches | Gaps | | ----------- |:---------:|:----------:|:----------:|:----------:|:----------:|:----------:|:----------:| | HM587118 | 489 | YP003848705.1 |M|glycoprotein|100|0|0| |MG972973|3591|YP003848705.1|M|glycoprotein|99.3|8|0| |DQ380221|3591|YP003848705.1|M|glycoprotein|99|4|7|0| |AF134496|738|YP003848705.1|M|glycoprotein|98.8|3|0| |DQ380222|3591|YP003848705.1|M|glycoprotein|99.2|9|0| |EU574086.1|795|YP003848706.1|S|non-structural protein|97.4|7|0| |EU574086.1|735|YP003848707.1|S|nucleocapsid|99.6|1|0| |RVFVNamibia2011MT561463NAM2011|3558|YP003848705.1|M|glycoprotein|99.2|9|0| |DQ380218|3591|YP003848705.1|M|glycoprotein|99.5|6|0| |HM587108|489|YP003848705.1|M|glycoprotein|100|0|0| |DQ380189|3591|YP003848705.1|M|glycoprotein|98.9|13|0| |HM587125|489|YP003848705.1|M|glycoprotein|99.4|1|0| |HM587045|489|YP003848705.1|M|glycoprotein|100|0|0|

Web application.

The tool is also implemented as a web application at https://www.genomedetective.com/app/typingtool/rvfv/

Pipeline Summary

By default, the pipeline currently performs the following:

• Classification of query sequence(s) (diamond)
• Phylogenetic typing (iqtree)

Credits

rvfvtyping was originally written by John Juma.

We thank the following people for their extensive assistance in the development of this pipeline: - Vagner Fonseca - Peter Van Heusden

License

rvfvtyping is free software, licensed under GPLv3.

Issues

Please report any issues to the issues page.

Contribute

If you wish to fix a bug or add new features to the software we welcome Pull Requests. We use GitHub Flow style development. Please fork the repo, make the change, then submit a Pull Request against out master branch, with details about what the change is and what it fixes/adds. We will then review your changes and merge them, or provide feedback on enhancements.

Citations

rvfvtyping pipeline uses the following software:

Buchfink, B., Xie, C., & Huson, D. H. (2015). Fast and sensitive protein alignment using DIAMOND. Nature Methods, 12(1), 5960. https://doi.org/10.1038/nmeth.3176

Guindon, S., & Gascuel, O. (2003). A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Systematic Biology, 52(5), 696704. https://doi.org/10.1080/10635150390235520

Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q., & Vinh, L. S. (2018). UFBoot2: Improving the Ultrafast Bootstrap Approximation. Molecular Biology and Evolution, 35(2), 518522. https://doi.org/10.1093/molbev/msx281

Huelsenbeck, J. P., & Ronquist, F. (2001). MRBAYES: Bayesian inference of phylogenetic trees. Bioinformatics, 17(8), 754755. https://doi.org/10.1093/bioinformatics/17.8.754

Katoh, K. (2002). MAFFT: A novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30(14), 30593066. https://doi.org/10.1093/nar/gkf436

Larkin, M. A., Blackshields, G., Brown, N. P., Chenna, R., McGettigan, P. A., McWilliam, H., Valentin, F., Wallace, I. M., Wilm, A., Lopez, R., Thompson, J. D., Gibson, T. J., & Higgins, D. G. (2007). Clustal W and Clustal X version 2.0. Bioinformatics, 23(21), 29472948. https://doi.org/10.1093/bioinformatics/btm404

Vilsker, M., Moosa, Y., Nooij, S., Fonseca, V., Ghysens, Y., Dumon, K., Pauwels, R., Alcantara, L. C., Vanden Eynden, E., Vandamme, A.-M., Deforche, K., & de Oliveira, T. (2019). Genome Detective: An automated system for virus identification from high-throughput sequencing data. Bioinformatics, 35(5), 871873. https://doi.org/10.1093/bioinformatics/bty695

Yu, G., Smith, D. K., Zhu, H., Guan, Y., & Lam, T. T.-Y. (2017). ggtree: An r package for visualization and annotation of phylogenetic trees with their covariates and other associated data. Methods in Ecology and Evolution, 8(1), 2836. https://doi.org/10.1111/2041-210X.12628

An imagemagick-like frontend to Biopython SeqIO seqmagick