Pneumo-Typer is an integrated tool for simultaneous identification of serotype, sequence type, and capsule genotype in Streptococcus pneumoniae. It is freely available at https://www.microbialgenomic.cn/Pneumo-Typer.html and https://github.com/Xiangyang1984/Pneumo-Typer under an open-source GPLv3 license.
Pneumo-Typer is a Perl script that doesn't need compilation. But before running, Pneumo-Typer needs to pre-install prodigal, blat, blast, and several Perl modules. There are two ways to install the Pneumo-Typer.
We have built a bioconda package for Pneumo-Typer v1.0.3. Users are recommended to install the conda, and then install this package simply with the following command:
$ conda install -c bioconda pneumo-typer
Once the installation is finished, the absolute paths for blat, prodigal, blastn and makeblastdb have been auto-configured well for pneumo-typer.pl, so users should be able to run Pneumo-Typer.
Installation of Pneumo-Typer can be accomplished by downloading the code (at https://www.microbialgenomic.cn/Pneumo-Typer.html and https://github.com/xiangyang1984/Pneumo-Typer) and then following the steps below.
Download Pneumo-Typer, and put the Pneumo-Typer directory into your PATH with the following command：
```
$ wget -c https://www.microbialgenomic.cn/gz/pneumo-typer-v1.0.3.tar.gz (***Recommended to use***)
$ git clone https://github.com/xiangyang1984/Pneumo-Typer.git (***Not recommended to use***)
***In case of downloading Source Code through git, two large-size files of Pneumo-Typer need to be downloaded manually.***.
* cgMLST_profiles
$ wget -c https://www.microbialgenomic.cn/gz/cgMLST_profiles.tar.gz && tar -xzvf cgMLST_profiles.tar.gz -C path_to_Pneumo-Typer/ST_tool/database/cgmlst
* cgMLSA_loci.fas
$ wget -c https://www.microbialgenomic.cn/gz/cgMLSA_loci.fas.tar.gz && tar -xzvf cgMLSA_loci.fas.tar.gz -C path_to_Pneumo-Typer/ST_tool/database/cgmlst/cgMLSA_loci
$ export PATH=/path/to/Pneumo-Typer/:$PATH
```
Pneumo-Typer requires Perl as well as Perl modules including GD; GD::SVG, SVG; threads, File::Basename, FindBin, File::Spec, lib, Getopt::Long, Math::BigFloat, Storable, vars, Bio::SeqIO, Bio::Tree::NodeI, Bio::TreeIO.
These modules can be installed with cpan using:
$ sudo cpan install GD GD::SVG SVG threads File::Basenamey FindBin lib Getopt::Long Math::BigFloat Storable vars BioPerl
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
Once Pneumo-Typer installation is finished, a small dataset in the ./Test_data directory can be used to test whether Pneumo-Typer (pneumo-typer.pl) can run on your system (Linux/MacOS) successfully or not using the command as below:
$ perl pneumo-typer.pl -Ts T
Test-step1: Checks for pneumo-typer dependencies...
################################################################
***GD Version 2.71 ok.
***GD::SVG Version 0.33 ok.
***SVG Version 2.84 ok.
***threads Version 2.15 ok.
***File::Basename Version 2.85 ok.
***FindBin Version 1.51 ok.
***lib Version 0.63 ok.
***Getopt::Long Version 2.49 ok.
***Math::BigFloat Version 1.999806 ok.
***Storable Version 3.15 ok.
***vars Version 1.03 ok.
***File::Spec Version 3.75 ok.
***Bio::SeqIO Version 1.007002 ok.
***Bio::Tree::NodeI Version ok.
***Bio::TreeIO Version 1.007002 ok.
!!!Ok, all dependencies Perl modulers are installed*
----------------------------------------------------------------
Checking for makeblastdb ... OK, makeblastdb is installed at: /miniconda3/bin//makeblastdb
Checking for blastn ... OK, blastn is installed at: /miniconda3/bin//blastn
Checking for prodigal ... OK, prodigal is installed at: /miniconda3/bin//prodigal
Checking for blat ... OK, blat is installed at: /miniconda3/bin//blat
################################################################
Test-step2: Begin test pneumo-typer.pl...
################################################################
Fri Jul 21 19:56:13 2023: pneumo-typer.pl start...
STEP-1: Dealing with genomes extract genome sequence, gene sequences and gene feature table (TFT); annotate genome which has no annotation information using prodigal>
Annotating genome using prodigal: 50%...100%...done
Genenucleotide_TFT_extract_percent: 50%...100%...done
STEP-2: Determining the sequence type (ST/cgST)
STEP-2.1: MLST analysis
Blastn_percent: 50%...100%...done
STEP-2.2: cgMLST analysis
Blat_percent: 50%...100%...done
STEP-3: Predicting serotype
Blastn_percent: 50%...100%...done
Process data and obtain serotype...done
STEP-4: Output sequence type and serotype results
STEP-5: Heatmaping the cps gene distribution in genomes
STEP-6: Visualizing the cps gene cluster in each genome
Fri Jul 21 20:07:30 2023: done!
################################################################
Ok, Pneumo-Typer works successfully!
Before using Pneumo-Typer, users should update the mlst dataset for ST analysis and/or cgmlst dataset for cgST analysis (optinal). It is very simple to use Pneumo-Typer. First, prepare input data, at least containing a Genbank_file_directory (containing files in GenBank format, FASTA format, or a combination of both); then, run Pneumo-Typer like this "perl pneumo-typer.pl -d Genbank_file_directory".
Before using Pneumo-Typer, users should run path_to_update_mlstdb_cgmlstdb.pl to update the mlst and/or cgmlst dataset (optinal).
$ perl path_to_update_mlstdb_cgmlstdb.pl -m T -c F
Update both mlst dataset and cgmlst dataset with 4 parallels (note: sets more parallels may lead to download failtures for some genes) using the following command
$ perl path_to_update_mlstdb_cgmlstdb.pl -m T -c T -t 4
Here, we used 18 genomes as an example to show how to use Pneumo-Typer. 18 genomes are under a directory named "18_genomes_dir"(downoload 18_genomes_dir.tar.gz).
$ 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 (about 3 min for one genome), 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
*Create a figure of the genetic organization of cps gene using the following command:
$ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rf T -tree path_to_18_genome_tree.nwk
Example 3: A two-column tab-delimited text file is used to sort genomes from up to down according to users' requirement
Here, we provided a srf file named "18_genome_order.txt" that orders the maps by serotypes. For example, users can reorder genomes in the genetic organization of the cps cluster.
$ 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-typer_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 needs about 3 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 organization 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 associate the genomes with their phylogeny. Meanwhile, Pneumo-Typer will output a file named "temp_strain_reorder_file-svg.txt", which contains the order information of genomes in the tree from up to down. It should be noted that all node names in the provided tree must completely match the input file names of all genomes. This parameter can only function when -Rh or -Rf set to "T".
-srf, --strain_reorder_file
A two-column tab-delimited text file is used to sort genomes from up to down according to users' requirements. 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 strain names must completely match the input file names of all genomes. This parameter can only function when -Rh or -Rf set to "T".
-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.
Dr. Xiangyang Li (E-mail: lixiangyang@fudan.edu.cn, lixiangyang1984@gmail.com), Kaili University; Bacterial Genome Data mining & Bioinformatic Analysis (https://www.microbialgenomic.cn/).
Copyright 2024, Xiangyang Li. All Rights Reserved.