This pipeline is written specifically for annotating the bacteria whole genome sequences (WGS). The pipeline handles multiple operations that are necessary for bacterial genome analysis. Including:

1. Annotating bacterial WGS
2. Constructing a pangenome for bacterial WGS dataset
3. Identify core and accessory loci for bacterial WGS dataset
4. Produce core gene concatenation alignment (with & without recombination detection)
5. Identify potential recombination regions (recent & ancestral) - (WGS wise & Per-gene)
6. Identify SNPs from conserved regions of the bacterial genomes
7. Reconstruct Phylogeny of input dataset (Maximum Liklihood)
8. Add Annotation to alignment and ML trees taxa (designed for BEAST Analysis)

1. Install conda (Python3) in your local computer or on the computing cluster. Detailed instruction can be find here

2. make a working directory

   mkdir {BactPrep_dir}*
   
   cd {BactPrep_dir}
   

   * this name can change base on your project

3. clone the repository into local working directory

   git clone https://github.com/rx32940/BactPrep.git
   

4. If first time using the pipeline

   cd BactPrep 
   
   conda create -n BactPrep python=3 mamba -c conda-forge -y
   
   conda activate BactPrep 
   
   # if this step is not complete, set channel priirity in conda env to flexible with command: 
   # conda config --set channel_priority true
   mamba install --file workflow/env/install.yaml  
   
   source INSTALL.sh 
   
   

   * this name can change base on your project

• • 4.1) if used the pipeline before or has matlab runtime R2016b (MCR) AND fastGear executable installed on the machine, use flag --mcr_path and --fastgear_exe to specify the absolute path to MCR and fasrGear executable. IF these two software were installed during previous use of BactPrep. you can find them in the resources folder from the previous download (please see example #6 below for detail).

5. You are now good to go! RUN:

   start_analysis.py ALL(coreGen/wgsRecomb/panRecomb)
   

   OR

   python start_analysis.py ALL(coreGen/wgsRecomb/panRecomb)
   

6. after running all your analysis, deactivate the env

   conda deactivate
   

218 Streptococcus pneumoniae PMEN1 WGS assemblies collected from the year 1984 - 2008 from 22 unique countries globally - The sample dataset can be downloaded to your work directory by:

mkdir -p $INPATH/assemblies

cd $INPATH/assemblies

zenodo_get -d 10.5281/zenodo.5603335

rm $INPATH/assemblies/md5sums*

Reference genome for Streptococcus pneumoniae PMEN1 can be downloaded from NCBI: Streptococcus pneumoniae ATCC 700669 (firmicutes)

cd $INPATH/

wget https://ftp.ncbi.nlm.nih.gov/genomes/all/GCF/000/026/665/GCF_000026665.1_ASM2666v1/GCF_000026665.1_ASM2666v1_genomic.fna.gz

gunzip GCF_000026665.1_ASM2666v1_genomic.fna.gz

ALL: this module will attempt to run wgsRecomb, coreGen, and coreRecomb module - All options required for these three modules are also required for ALL module

wgsRecomb: detect recombination from WGS alignment

coreGen: construct bacteria pangenome

panRecomb: will attempt to detect recombination for each gene in the all genes in the pangenome individually - predict recombinaiton among lineages detected by BAPs (can also provide your own lineage) - this module use gene loci detected by Roary, thus will also run module coreGen - please use geneRecomb module for individual gene/alignment of interest

geneRecomb: will detect recombination from a gene/alignment interested

coreRecomb: will dect recombinations only from the core genes detected by coreGen module (Roary) - this is part of the ALL module - this module use gene loci detected by Roary, thus will also run module coreGen - will mask detected recombination region, and call SNPs from conserved region of core genome alignment - recombinations were detected for each gene individually - will also reconstruct phylogeny for the dataset based on the core clonal SNPs.

usage: start_analysis.py MODULE [options]

Please always specify the program to use in the first argument, or the whole pipeline will attemp to run

positional arguments:
  {ALL,wgsRecomb,coreGen,coreRecomb,panRecomb,geneRecomb}
                        Specify the module you would like to run

optional arguments:
  -h, --help            show this help message and exit

general arguments:
  -i , --input          path to input dir with assemblies
  -p , --name           provide name prefix for the output files
  -t , --thread         num of threads
  -o , --output         path to the output directory

arguments for if you would like to add metadata to output:
  -M, --addMetadata     must have the flag specify if want to allow annotation
  -a , --annotate       path to a csv file containing sample metadata
  -s , --sample         integer indicates which column the sample name is in the metadata csv file
  -m , --metadata       metadata chosen to annotate ML tree/alignment after the sample name

arguments for wgsRecomb module:
  -r , --ref            reference (required for wgsRecomb module)
  -v , --phage          phage region identified for masking (bed file)
  -G , --gubbins        any additional Gubbins arguments (please refer to Gubbins manual)

arguments for coreGen module:
  -g , --gff            path to input dir with gff (this can replace input assemblies dir in coreGen module Must be gff3 files)
  -c , --core           define core gene definition by percentage for coreGen module (default=99)
  -k , --kingdom        specify the kingom of input assemlies for genome annotation (default=Bacteria)
  -R , --roary          any additional roary arguments (please refer to Roary manual)

arguments for all three fastGear modules (coreRecomb, panRecomb, geneRecomb):
  --mcr_path            path to mcr runtime (need to install before use any of the fastGear module
  --fastgear_exe        path to the excutable of fastGear
  --fg , --fastgear_param 
                        path to fastGear params

arguments for geneRecomb module:
  -n , --alignment      input alignment (either -n/-fl is required for geneRecomb module)
  -fl , --alnlist       input alignment list with path to gene alignments (either -n/-fl is required for geneRecomb module)

Enjoy the program! :)

Run: start_analysis.py ALL(coreGen/wgsRecomb/panRecomb)

1) Get Start - How to run ALL Module if you would like to run "wgsRecomb", "coreGen", and "coreRecomb" modules all together, you can just use the "ALL" module. Note: a reference genome (-r) is necessary to run "wgsRecomb" module

EXAMPLE:

start_analysis.py ALL -p PMEN1.dated \
-o $OUTPATH \
-i $INPATH/assemblies \
-r $INPATH/GCF_000026665.1_ASM2666v1_genomic.fna

1.1) If you already have gff files obtained from previous analysis, gff dir can be used as input for "coreGen module". This will saves a lot time

start_analysis.py ALL -p PMEN1.dated \
-o $OUTPATH \
-t 10 \
-g $INPATH/gff \ # gff dir as input
--mcr_path {path_to_previous_BactPrep_folder}/resources/mcr \ # absolute path to mcr R2016b
--fastgear_exe /home/user/SOFTWARE/fastGEARpackageLinux64bit # absolute path to fastGear excutable

2) Obtain Annotated Outputs if you would like to obtain annotated phylogenies and alignments, please provide a CSV file with annotation of every isolates. Flag -M must be specified for annotation. -a is the path to the CSV metadata file. -s allows the you to specify the index of the column matches with the input assemblies' file names, default is 1. -m asks for the column names of the metadata you would like to add for annotations (comma separated).

EXAMPLE CSV File:

EXAMPLE:

start_analysis.py ALL -p PMEN1.dated \
-o $OUTPATH \
-i $INPATH/assemblies \
-r $INPATH/GCF_000026665.1_ASM2666v1_genomic.fna \
-M \
-a $INPATH/PMEN1.dated.metadata.csv \
-s 1 \
-m Year,Country

3) IF you would only like to run "wgsRecomb" please keep in mind, a reference genome must provided by user."wgsRecomb" module will call snps from the reference genome for each input WGS assemblies, and combine them into a multiple sequence alignment using Snippy, where genome regions shared by all isolates in the input dataset will be extracted. Gubbins will take the Snippy input to detect recombination regions from the multi-sequence alignment. At end of the pipeline, SNPs outside of the recombination regions will be used to reconstruct the input dataset's phylogeny with IQTree. Annotation will be added to phylogenies and SNPs alignments if a metadata file is provided by user (please see example 2 for details).

EXAMPLE:

start_analysis.py wgsRecomb -p PMEN1.dated \
-o $OUTPATH \
-i $INPATH/assemblies \
-r $INPATH/GCF_000026665.1_ASM2666v1_genomic.fna 

4) IF you would only like to run "coreGen" a reference file would be required for this module. All input WGS assemblies will be annotated by Prokka. Using prokka's gene annotations, Roary will 1) reconstruct the pangenome of the input dataset, and 2) identify core genes shared by 99% (this can be adjust by user by -c flag) of the isolates in the input dataset. Roary will also provide a core gene concatenation alignment, which will be used for phylogeny reconstruction using IQTree at end of the pipeline. Annotation will be added to phylogenies and SNPs alignments if a metadata file is provided by user (please see example 2 for details).

EXAMPLE:

start_analysis.py wgsRecomb -p PMEN1.dated \
-o $OUTPATH \
-i $INPATH/assemblies 

5) IF you would only like to run "coreRecomb" pipeline implemented in the "coreGen" module will run first. "coreRecomb" module will identify homologous recombinaition from every core gene identified by Roary. The identified recombination regions will be masked in the gene alignments before all core genes' masked alignments are concatenated into a super-gene alignment. core SNPs outside of the recombination regions will be called, SNPs outside of the recombination regions will be used to reconstruct the input dataset's phylogeny with IQTree. Annotation will be added to phylogenies and SNPs alignments if a metadata file is provided by user (please see example 2 for details).

EXAMPLE:

start_analysis.py coreRecomb \
-p PMEN1.dated \
-o $WORKPATH -i $WORKPATH/assemblies \
-r $WORKPATH/GCF_000026665.1_ASM2666v1_genomic.fna \
-t 10 \
-M \
-a $WORKPATH/PMEN1.dated.metadata.csv \
-m Year,Country 

6) IF matlab runtime (MCR) version R2016a is installed or this is not the first time you are running this pipeline. if you have already installed MCR R2016a and fastGear executable before on your machine, or you have already installed these two dependencies the previous times you were using BactPrep. You can use flag --mcr_path and --fastgear_exe to avoid installing these two dependencies again. you don't need to run INSTALL.sh script again if these two scripts is already installed, but a conda env still need to be created and activated to run BactPrep pipeline

EXAMPLE:

conda env create -f workflow/env/install.yaml -n BactPrep

conda activate BactPrep

start_analysis.py panRecomb -p PMEN1.dated_fastGear_pan \
-o $OUTPATH \
-t 10 \
-i $INPATH/assemblies \
--mcr_path {path_to_previous_BactPrep_folder}/matlab/v901 \
--fastgear_exe {path_to_previous_BactPrep_folder}/fastGEARpackageLinux64bit 

7) IF you would like to inform wgsRecomb (gubbins) about already known phage region phage region can be provided to snippy before running gubbins. If you would like to provide known phage region while running gubbins, use -v or --phage to provide phage region in a BED file.

EXAMPLE:

start_analysis.py wgsRecomb \
-p PMEN1.dated \
-o $WORKPATH -i $WORKPATH/assemblies \
-r $WORKPATH/GCF_000026665.1_ASM2666v1_genomic.fna \
-v $WORKPATH/phage_region.bed

8) IF additional arguments need to be specificed for Roary and Gubbins when using "coreGEN", "wgsRecomb", or "ALL" module additional Roary and Gubbins arguments that is not specificed by BactPrep can be added by using the -R of -G flags, respectively. Dependencies used for these additional arguments need to be install by user.

SPACE is necessary at the beginning of the string

Example:

start_analysis.py ALL \
-p PMEN1.dated \
-o $WORKPATH -g $WORKPATH/gff \
-r $WORKPATH/GCF_000026665.1_ASM2666v1_genomic.fna \
-R " -r -y -iv 1.5"

5. If you have trouble installing fastGear with INSTALL.sh script. please follow the instruction below for installation.

   1. mcr has many versions, use the link to download the version compatible with fastGear:

      Download and install fastGear excutable: 1. change directory to: {absolute_path_to_BactPrep}/resources/mcr 2. you can download mcr provided by fastGear developers: https://users.ics.aalto.fi/~pemartti/fastGEAR/ wget --no-check-certificate https://users.ics.aalto.fi/~pemartti/fastGEAR/fastGEARpackageLinux64bit.tar.gz -P {absolute_path_to_BactPrep}/resources) 3. Unzip the downloaded file tar -zvxf fastGEARpackageLinux64bit.tar.gz

      Download and install Matlab Runtime:

      1. Download MCR zip provided by fastGear developers: wget https://users.ics.aalto.fi/~pemartti/fastGEAR/MCRInstallerLinux64bit.zip -P {absolute_path_to_BactPrep}/resources --no-check-certificate
      2. Unzip the downloaded file unzip MCRInstallerLinux64bit.zip
         1. or download version R2016a from MATLAB: https://www.mathworks.com/products/compiler/matlab-runtime.html
      3. change directory after unzip the downloaded file cd MCRInstallerLinux64bit
      4. install: ./install -destinationFolder {absolute_path_to_BactPrep}/resources/mcr/ -mode silent -agreeToLicense yes
         1. if you would like to install with a GUI interface, please allow X11 display at the terminial, do ./install, this will open the GUI installation, and will allow you to change the directory to install, please install to {absolute_path_to_BactPrep}/resources/mcr

   2. if you already have mcr (R2016a) on your machine (or used this pipeline before), you do not need to reinstall mcr, please specify the absolute path with --mcr_path flag, which leads to the absolute path of your installed mcr

      1. --mcr_path: ex. --mcr_path {absolute_path_to_BactPrep}resources/mcr/