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Pneumo-Typer User guide

     ● Installation
  
              ○ Installing the Pneumo-Typer via Conda
  
              ○ Installing the Pneumo-Typer from Source Code
  
              ○ Test the Pneumo-Typer with Example Data
  
     ● Usage
  
              ○ Running Pneumo-Typer
  
              ○ Detailed Explanations for Arguments in Pneumo-Typer  

      $ conda install -c bioconda pneumo-typer

 
Recommended to use: 
        $ wget -c https://www.microbialgenomic.cn/gz/pneumo-typer-v1.0.1.tar.gz

not recommended to use:
        $ git clone https://github.com/xiangyang1984/Pneumo-Typer.git

        In case of downloading Source Code through git, two large-size files of Pneumo-Typer need to be downloaded manually.
        * cgMLST_profiles
            $ wget -c -O path_to_Pneumo-Typer/ST_tool/database/cgmlst/cgMLST_profiles https://media.githubusercontent.com/media/Xiangyang1984/Pneumo-Typer/main/ST_tool/database/cgmlst/cgMLST_profiles?download=true
        * cgMLSA_loci.fas
            $ wget -c -O path_to_Pneumo-Typer/ST_tool/database/cgmlst/cgMLSA_loci/cgMLSA_loci.fas https://media.githubusercontent.com/media/Xiangyang1984/Pneumo-Typer/main/ST_tool/database/cgmlst/cgMLSA_loci/cgMLSA_loci.fas?download=true

       $ export PATH=/path/to/Pneumo-Typer/:$PATH

      $ sudo cpan install GD GD::SVG SVG threads File::Basenamey FindBin lib Getopt::Long Math::BigFloat Storable vars BioPerl

Step 3: Programs installation
Additional software dependencies for the Pneumo-Typer are as follows:

* makeblastdb and blastn 
Both of them come from NCBI BLAST+, available at https://ftp.ncbi.nlm.nih.gov/blast/executables/blast+/LATEST/

* prodigal
This software is available at https://github.com/hyattpd/Prodigal

* blat
This software is available at https://genome.ucsc.edu/FAQ/FAQblat.html

$ perl pneumo-typer -Ts T

    $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -t 10 -m T

Pneumo-Typer will perform serotype prediction, heatmap and figure creation, and ST analysis with 10 threads, output results as follows:
   * a. ST results
   * b. predicted serotype results
   * c. create three maps with the ST and predicted serotype and information showed
         *heatmap_gene.svg: a heatmap of the distribution of cps gene at gene level  
         *heatmap_class.svg: a heatmap of the distribution of cps gene at class level 
         *cps_cluster.svg: a figure showing the genetic organization of cps gene cluster

Setting "-c" to "T" will perform cgST analysis which takes quite a long time, and the cgST information will also be shown on maps.

Example 2: A Newick format tree file is used by Pneumo-Typer to automatically associate the distribution of cps gene and genetic organization of cps cluster with their phylogeny.

18_genome_tree.nwk: a tree file, it was a nwk format phylogenetic tree of 18 genomes using RaxML. 
 
*Create two heatmaps according to the distribution of the cps gene at the gene and class level using the following command:

    $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rh T -tree path_to_18_genome_tree.nwk 

    $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rf T -tree path_to_18_genome_tree.nwk

    $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rf T -srf path_to_18_genome_order.txt

    REQUIRED ARGUMENTS:
    ~~~~~~~~~~~~~~~~~~~
       -d, --genbank_file_directory
           A directory containing files in GenBank format, FASTA format, or a combination of both.                           
    OPTIONAL ARGUMENTS:
    ~~~~~~~~~~~~~~~~~~~
       -o, --output_directory
           An output directory holding all the generated files by pneumo-typer.pl. if this option is not set,  a directory named "pneumo-pyper_workplace" will be created in the bin directory from where pneumo-typer.pl was invoked.
       -t, --multiple_threads
           Set thread number (Default: 1)
       -Ss, --skip_sequence_processing 
           Skip the process of sequence processing (STEP-1) (Default: F).  
       -hgc, --homologous_gene_cutoff
           Set E-value, Identify, Coverage (Query and Subject), Match_length (alignment length) cutoff in Blastn analysis (default: E-value=1e-5, Identify=70, Coverage=95, Match_length=100).
       -Sb, --skip_blastn
           Skip the process of doing blastn during serotype analysis.
       -p, --prodigal_annotation
           Annotate all genomes using prodigal. 
       -m, --mlst
           Perform mlst analysis (Default: T). 
       -c, --cgmlst
           Perform cgmlst analysis. It need >10 mins for one genome (Default: F).
       -Rh, --recreate_heatmap                             
           Re-create the heatmap of cps gene distribution in genomes (Default: F). At this step, users can add a parameter "phylogenetic_tree" or "strain_reorder_file". 
       -Rf, --recreate_figure
           Re-create the figure of the genetic organlization of cps gene cluster for genomes (Default: F). At this step, users can add a parameter "phylogenetic_tree" or "strain_reorder_file".
       -tree, --phylogenetic_tree
           A Newick format tree file is used by Pneumo-Typer to automatically accociate the genomes with their phylogeny. Meanwhile, Pneumo-Typer will output a file named "temp_strain_reorder_file", which contains the order information of genomes in tree from up to down. It should be noted that all nodes name in provided tree must completely match with the genbank files name of all genomes.
       -srf, --strain_reorder_file
           A two-column tab-delimited text file is used to sort genomes from up to down accoding to users requirement. Each row must consist of a strain name followed by the numerical order that is used for sorting genomes. It should be noted that all strains name must completely match with the genbank files name of all genomes.
       -Ts, --test
           Run pneumo-typer using Test_data as input to check whether Pneumo-Typer is installed successfully (Default: F).
       -V, --version
           The version of Pneumo-Typer.
       -h, --help
           Show this message.

COPYRIGHT

Dr. Xiangyang Li (E-mail: lixiangyang@fudan.edu.cn, lixiangyang1984@gmail.com), Kaili University; Bacterial Genome Data mining & Bioinformatic Analysis (http://www.microbialgenomic.cn/).

Copyright 2023, Xiangyang Li. All Rights Reserved.

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