Prophage Sequence Typing pipeline is developed in R language and is a R wrapper for multiple functions; This pstR is based on a previous PST bash pipeline: author='duceppemo' version='0.2.0'
In a terminal, conda create a "phage_typing" environment and install three software fastp(v0.20.1), spades(v3.13.2) and cd-hit(v4.8.1), or qiime2 if it is possible with the environment compatibility.
conda create –n phage_typing –c bioconda fastp spades cd-hit qiime2
ALTERNATIVELY: If qiime requires a different version python, which is incompatible with "phage_typing" environment, the qiime environment can be created separately
conda create -n qiime
conda install -c qiime2 qiime2
Two more scripts named "checkPhasterServer.py" and "cdHitClstr2table.pl" need to be downloaded from the folder named "scripts". The PATH containing this two scripts need to be called in R envrironment as puting as parameters in two steps of this pipeline.
Download prophageTypingR_0.1.0.tar.gz from
https://github.com/susanruimingao/pstR.git
Go to work directory containing raw reads, and the reads name is preferred in "_R1.fastq.gz"/"_R2.fastq.gz"; otherwise specify in the command line.
Go to R environment and install three required packages, which will be needed to be installed once. other packagesthe above downloaded and four
R
install.packages("parallel");
install.packages("crayon");
install.packages("stringr");
install.packages("seqinr");
library(parallel)
library(crayon)
library(stringr)
library(seqinr)
Every time for enterring the R envrionment, the above downloaded prophageTypingR_0.1.0.tar.gz package need to be installed
install.packages("/home/CFIA-ACIA/gaoru/R_package/prophageTypingR_0.1.0.tar.gz", repos = NULL, type="source")
To run the pipeline steps until submitting to PHASTER, using general function: including trim reads, spades assembly and submit the assemblies to PHASTER server The default RawReads format suffixNameR1 = "_1.fastq.gz",suffixNameR2 = "_2.fastq.gz". Otherwise, specifiy as following:
prophageTypingR::trimAssembleSubmit(inputDir = "rawdata", suffixNameR1 = "_R1.fastq.gz",suffixNameR2 = "_R2.fastq.gz")
For continous run from last step: after submitting the assemlies to PHASTER, to check the status of PHASTER server running:
prophageTypingR::CheckPhasterServer(path = "/home/CFIA-ACIA/gaoru/bin/phage_typing/checkPhasterServer.py")
ALTERNATIVELY: If you want to start wit some completed genomes, specify the sffix type, becasue the defaulst ones are: inputDir = "spadesOut", suffix = "_assembly_filter2000.fasta", outputDir = "PHASTEROut"
prophageTypingR::submit_to_PHASTER (inputDir = "12AdjameGenome", suffix = ".fasta", outputDir = "PHASTEROut_12AdjGenome")
After downloading zip files from PHASTER server
##(Option 1): qiime is installed in the "phage_typing" environment One step of running to get the final phylogenetic tree file (including retrieve phage .fasta sequences, clustering and create phylogenetic tree with qiime)
prophageTypingR::ClusterRunQiime()
##(Option 2): if the qiime environment is installed individually.
under phage_typing environment
prophageTypingR::extract_fasta(inputDir = "PHASTEROut", outputDir = "fastaRetrieve")
prophageTypingR::cluster_sequences(inputFile = "./extractFasta/all_phage.fasta", c = 0.99, s = 0.99, outputDir = "./extractFasta/clusterSeqs_99_99", path = "/home/CFIA-ACIA/gaoru/bin/phage_typing/cdHitClstr2table.pl")
Under qiime environment:
prophageTypingR::runQiime(inputFile = "./extractFasta/clusterSeqs_99_99/phage_clustered_c0.99_s0.99.fasta.clstr", sampleList = "./extractFasta/sampleList.txt", path = "YOUR_OWN_PATH_to../cdHitClstr2table.pl")
***The finall created .tree file is the desired output
OPTIONAL: the individual functions are also available if you want to run each of them separately:
prophageTypingR::trimReads(inputDir = "rawdata", suffixNameR1 = "_R1.fastq.gz",suffixNameR2 = "_R2.fastq.gz" );
prophageTypingR::assemblySpades()
prophageTypingR::submit_to_PHASTER()
prophageTypingR::CheckPhasterServer(path = "YOUR_OWN_PATH_to../checkPhasterServer.py")
prophageTypingR::extract_fasta()
prophageTypingR::cluster_sequences(inputFile = "./extractFasta/all_phage.fasta", c = 0.99, s = 0.99, outputDir = "./extractFasta/clusterSeqs_99_99")
prophageTypingR::create_biom_table(path = "YOUR_OWN_PATH_to../cdHitClstr2table.pl")
prophageTypingR::biom_convert()
prophageTypingR::beta_diversity()
prophageTypingR::neighbor_joining()