SARS-CoV-2 Ratio of Homoplasic Offspring (RoHO)

This repository contains scripts and command lines used to compute the RoHO index of a set of high-quality filtered homoplasies. They were used in the publication

No evidence for increased transmissibility from recurrent mutations in SARS-CoV-2
Lucy van Dorp*, Damien Richard*, Cedric CS. Tan, Liam P. Shaw, Mislav Acman, François Balloux
*contributed equally
https://doi.org/10.1101/2020.05.21.108506

Homoplasies are mutations that emerged repeatedly and independently. They are good candidates for sites under natural selection. In our study, we used a phylogenetic index to assess whether particular homoplasic mutations increase transmissibility of SARS-CoV-2. To do so, we quantified the relative number of descendants in sister clades with and without a specific allele. This github repository contains the code used to do so.

The RoHO index is the ratio of the number of descendents of sister clades with and without a specific mutation over all independent emergences of a homoplasic allele in a phylogeny.

The script 'Main_homoplasy.sh" is to be run in a unix command line step by step to ensure proper functioning. You are likely to have to make some modifications to it in order to modify paths etc.

** This script is very likely to crash as it was not developped for general use but for the study of one particular dataset **

Inputs

• A fasta reference genome (not provided, Wuhan-Wu-1 download here :https://www.ncbi.nlm.nih.gov/search/api/download-sequence/?db=nuccore&id=NC_045512.2)
• A list of high-quality homoplasy positions (provided)
• Homoplasyfinder Consistency Index Report (provided, output file of Homoplasyfinder)
• Annotated tree file (not provided due to copyright issues, output file of Homoplasyfinder)
• Alignement file (not provided due to copyright issues, fasta file of aligned sequences from Gisaid)

Outputs

• Outputs several figures, tables and statistics

Dependencies

Perl core
ape R package
phangorn R package
reshape2 R package
ggplot2 R package
doParallel R package (optional, needed for parallelization)
foreach R package (optional, needed for parallelization)

This is a two step pipeline :

First step in bash / perl:

Main_homoplasy.sh

From a filtered homoplasy list, this script :

• Converts a raw homoplasyfinder annotated tree to a filtered homoplasyfinder annotated tree
• Produces a matrix of alleles for each isolates at variable positions from an alignement fasta file

The matrix has the following form :

	Isolate_1	Isolate_2	Isolate_3	Isolate_4
1912	"ref"	"ref"	"not_ref"	"ref"
11083	"ref"	"not_ref"	"ref"	"undef"
23043	"ref"	"ref"	"not_ref"	"ref"

Second step in R:

Homoplasy_ratios.R

In the filtered phylogeny (tree file), for each filtered node of the phylogeny annotated by HomoplasyFinder as corresponding to an ancestor that acquired a homoplasy, this script counts the number of offsprings having "ref", "not_ref" or "undef" alleles based on the input matrix. To be considered, an internal node must meet the following criterion :

• No children nodes themselves annotated as carrying the homoplasy.
• Having at least two descendant tips of each allele
• An homoplasic position is only considered for RoHo score computation if at least n=3 or n=5 nodes satisfy the two first criteria.

Acknowledgements

This study was rendered possible only thanks to the very large number of scientists in originating and submitting labs who have readily made available SARS-CoV-2 assemblies to the research community. They are listed in originating-laboratories.txt and submitting-laboratories.txt files.

A test dataset with no copyright issue will hopefully be provided in a few days in order to get the scripts running