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Pneumo-Typer: a high-throughput capsule genotype visualization tool with integrated serotype and sequence type prediction for Streptococcus pneumoniae
Pneumo-Typer is a high-throughput capsule genotype visualization tool with integrated serotype and sequence type prediction for 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. When you use Pneumo-Typer, please cited:
Xiangyang Li, Yaoyao Zhu, Zilin Yang, Yanzi Li, Guohui Zhang, Xiangyu Wang, Xuan Zhao, Yinyan Huang, Huajie Zhang, Bingqing Li, Zhongrui Ma. Pneumo-Typer: a high-throughput capsule genotype visualization tool with integrated serotype and sequence type prediction for Streptococcus pneumoniae, biorxiv, 2025, https://doi.org/10.1101/2025.02.13.638184. [download pdf]
Software download: pneumo-typer-v2.0.1.tar.gz
1. Installation
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.
1-1.1 Installing the Pneumo-Typer via Conda
We have build a bioconda package for Pneumo-Typer. Users are recommended to install the conda, then to 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.
1-1.2 Installing the Pneumo-Typer from Source Code
Pneumo-Typer is available at https://www.microbialgenomic.cn/Pneumo-Typer.html and https://github.com/Xiangyang1984/Pneumo-Typer. Installation Pneumo-Typer can be accomplished by downloading the code and then following the steps below.
Step 1: Download source code
Download Pneumo-Typer, and put the Pneumo-Typer directory into your PATH with the following command：
Recommended to use:
$ wget -c https://www.microbialgenomic.cn/gz/pneumotyperData/pneumo-typer-v2.0.1.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 https://www.microbialgenomic.cn/gz/pneumotyperData/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/pneumotyperData/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
Step 2: Perl modules installation
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
1-1.3 Test the Pneumo-Typer with Example Data
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:
$ pneumo-typer -Ts T (installed via conda)
or $ perl path_to_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 seqeunces and gene feature table (TFT);
annnotate genome which has no annotation infotmation using prodigal>
Annotating genome using prodigal: 20%...40%...60%...80%...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!
2. Usage
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".
2-2.1 Update mlst and/or cgmlst dataset
Before using Pneumo-Typer, users should run path_to_update_mlstdb_cgmlstdb.pl to update the mlst and/or cgmlst dataset (optinal).
Update only mlst dataset using the following command
$ update_mlstdb_cgmlstdb -m T -c F (installed via conda)
or $ perl path_to_update_mlstdb_cgmlstdb.pl -m T -c F
Update both mlst dataset and cgmlst dataset using the following command with 4 parallels
$ update_mlstdb_cgmlstdb -m T -c T -t 4 (installed via conda)
or $ perl path_to_update_mlstdb_cgmlstdb.pl -m T -c T -t 4
2-2.2 Running Pneumo-Typer
Here, we used 18 genomes as an example to show how to use Pneumo-Typer. 18 genomes are under a directory named "18_genomes"(downoload 18_genomes.tar.gz).
Example 1: Run Pneumo-Typer is an easy task using the following command
$ pneumo-typer -d path_to_18_genomes_dir -t 10 -m T (installed via conda)
or $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -t 10 -m T
Pneumo-Typer will perform serotype prediction, capsule genetype assignment, ST analysis, map creation using 10 threads, output results as follows:
* a. Serotype.out: a table shows predicted serotype results
* b. capsule_genotype.out: a table shows capsule genetype results. Notes: the serotype labeled by CapT is the one with the highest number of matching capsule genes (serotype alignment). The serotype output by the Pneumo-Typer is further refined against several serotype-specific markers (serotype refinement) based on the initial serotype alignment. Therefore, discrepancies may exist between the CapT-labeled serotype and the software-output serotype: the former reflects the capsule gene matching status, while the latter represents the true serotype.
* c. ST_out.txt: a table shows ST results
* d. cgST_out.txt: a table shows cgST results (Optinal, cgST is not carried out in this example)
* e. Serotype_CapT_ST.out: a table merging of predicted serotype, capsule genetype, and ST/cgST
* f. create three maps with the predicted serotype, capsule genetype, ST/cgST information showed
*heatmap_gene.svg: a heatmap of the distribution of cps gene at gene level (Figue 1)
*heatmap_class.svg: a heatmap of the distribution of cps gene at class level (Figue 2)
*cps_cluster.svg: a figure showing the genetic organization of cps gene cluster (Figue 3)
Setting "-c" to "T" will perform cgST analysis which takes quite a long time (about 3 mins for one genome), and the cgST information will also be shown on maps.
Figure 1. A heatmap dispalying the distribution of the cps gene at the gene level created by Pnuemo-Typer in Example 1
Figure 2. A heatmap dispalying the distribution of the cps gene at the class level created by Pnuemo-Typer in Example 1
Figure 3. A figure of the genetic organization of cps gene created by Pnuemo-Typer in Example 1
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_genomes_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:
$ pneumo-typer -d path_to_18_genomes_dir -Rh T -tree path_to_18_genomes_tree.nwk (installed via conda)
or $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rh T -tree path_to_18_genomes_tree.nwk
*Create a figure of the genetic organization of cps gene using the following command:
$ pneumo-typer -d path_to_18_genomes_dir -Rf T -tree path_to_18_genomes_tree.nwk (installed via conda)
or $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rf T -tree path_to_18_genomes_tree.nwk
Figure 4. A figure of the genetic organization of cps gene associated with phylogenetic tree by Pnuemo-Typer in Example 2
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_genomes_order.txt" that orders the maps by serotypes. For example, users can reorder genomes in the genetic organization of the cps cluster.
$ pneumo-typer -d path_to_18_genomes_dir -Rf T -srf path_to_18_genomes_order.txt (installed via conda)
or $ perl path_to_pneumo-typer.pl -d path_to_18_genomes_dir -Rf T -srf path_to_18_genomes_order.txt
2-2.3 Detailed Explanations for Arguments in Pneumo-Typer
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", 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.
COPYRIGHT
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—2027, Xiangyang Li. All Rights Reserved.
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