### Start STAMP for Visualization Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(San-Juan,-April-2019) Launch the STAMP application for visualizing PICRUSt2 output. This command is executed after installing STAMP on a local machine. ```bash stamp ``` -------------------------------- ### Install PaPaRa Source: https://github.com/picrust/picrust2/wiki/Pre-requisite-installations Install the PaPaRa tool. This includes installing the BOOST library if it's not already present, then extracting and building PaPaRa, and finally linking the executable to '~/bin'. ```bash sudo apt-get install libboost-all-dev ``` ```bash tar -xzf papara_nt-2.5-dev.tar.gz cd papara_nt-2.5-dev sh build_papara2.sh ln -s $PWD/papara ~/bin/ ``` -------------------------------- ### Install GAPPA Source: https://github.com/picrust/picrust2/wiki/Pre-requisite-installations Install the GAPPA tool by extracting the archive, compiling the source code, and creating a symbolic link to the '~/bin' directory. ```bash tar -xzf gappa-0.0.0-dev.tar.gz cd gappa-0.0.0-dev make ln -s $PWD/bin/gappa ~/bin/ ``` -------------------------------- ### Verify q2-picrust2 Plugin Installation Source: https://github.com/picrust/picrust2/wiki/Manually-install-QIIME-2-plugin Verify the successful installation of the q2-picrust2 plugin by checking its help description. ```bash qiime picrust2 --help ``` -------------------------------- ### Install PICRUSt2 from GitHub Source: https://github.com/picrust/picrust2/wiki/Testing-the-updated-PICRUSt2-database Clone the repository, create a conda environment, and install the package in editable mode. This is the recommended method for testing the latest version. ```bash git clone --single-branch -b PICRUSt2-v2.6.0 https://github.com/picrust/picrust2 mv picrust2 picrust2-v2.6.0 cd picrust2-v2.6.0 conda env create -n picrust2-v2.6.0 -f picrust2-env.yaml conda activate picrust2-v2.6.0 pip install --editable . ``` -------------------------------- ### Install q2-picrust2 Plugin Source: https://github.com/picrust/picrust2/wiki/Manually-install-QIIME-2-plugin Install the q2-picrust2 plugin into the current conda environment by navigating to the decompressed plugin folder and running pip install. ```bash pip install -e . ``` -------------------------------- ### Install Miniconda Source: https://github.com/picrust/picrust2/wiki/Pre-requisite-installations Download and install the latest Miniconda for Linux x86_64. Follow the on-screen prompts to complete the installation. ```bash wget https://repo.continuum.io/miniconda/Miniconda3-latest-Linux-x86_64.sh bash Miniconda3-latest-Linux-x86_64.sh (follow on-screen instructions when prompted,"enter", "yes", "yes") ``` -------------------------------- ### Install EPA-NG Source: https://github.com/picrust/picrust2/wiki/Pre-requisite-installations Install the EPA-NG tool by extracting the archive, compiling the source code, and creating a symbolic link to the '~/bin' directory. If 'make' fails, install necessary compilation packages. ```bash tar -xzf epa-ng-0.2.1-beta-dev.tar.gz cd epa-ng-0.2.1-beta-dev make ln -s $PWD/bin/epa-ng ~/bin/ ``` ```bash sudo apt-get install autotools-dev libtool flex bison cmake automake autoconf ``` -------------------------------- ### Verify PICRUSt2 Installation Source: https://github.com/picrust/picrust2/wiki/Installation Runs the test suite for PICRUSt2 to verify that the installation was successful. This command should be run from the main PICRUSt2 source directory. ```bash pytest ``` -------------------------------- ### Install PICRUSt2 on Mac (M1/M2/M3) Source: https://github.com/picrust/picrust2/wiki/Installation Installs PICRUSt2 on Macs with M1/M2/M3 processors by creating a specific 64-bit macOS environment with Python 3.10, then activating it and installing from source. This workaround addresses known compatibility issues. ```bash wget https://github.com/picrust/picrust2/archive/v2.6.3.tar.gz tar xvzf v2.6.3.tar.gz cd picrust2-2.6.3/ mamba env create -f picrust2-env.yaml --platform osx-64 python=3.10 mamba activate picrust2 pip install --editable . ``` -------------------------------- ### Bacterial RAxML Info File Example Source: https://github.com/picrust/picrust2/wiki/Creating-the-PICRUSt2-database An example of a manually created RAxML info file for bacteria, derived from a RAxML log. This shows the specific parameters and values for bacterial datasets. ```text This is RAxML version 7.7.2 released by Alexandros Stamatakis on July 31 2013. This is a RAxML_info file from an --evaluate run, manually reformatted Partition: 0 Alignment Patterns: 1580 Name: No Name Provided DataType: DNA Substitution Matrix: GTR RAxML-NG was called at 24-Jan-2025 19:16:30 as follows: raxml-ng --evaluate --msa raxml/bacteria_raxml-check.raxml.reduced.phy --tree bac120_r220_reduced.tree --prefix raxml/bacteria_raxml --model GTR+G --threads 24 Base frequencies: 0.209478 0.233565 0.312776 0.244181 Inference[0]: Time 1617.080 CAT-based likelihood -0000, best rearrangement setting 5 alpha[0]: 1.000000 rates[0] ac ag at cg ct gt: 1.047126 2.926184 1.654626 0.822619 3.735054 1.000000 NOT conducting any final model optimizations on all 1 trees under CAT-based model .... Final GAMMA likelihood: -5582296.255705 ``` -------------------------------- ### Archિયલ RAxML Info File Example Source: https://github.com/picrust/picrust2/wiki/Creating-the-PICRUSt2-database An example of a manually created RAxML info file for archaea, derived from a RAxML log. This demonstrates how to populate the template with actual data. ```text This is RAxML version 7.7.2 released by Alexandros Stamatakis on July 31 2013. This is a RAxML_info file from an --evaluate run, manually reformatted Partition: 0 Alignment Patterns: 1409 Name: No Name Provided DataType: DNA Substitution Matrix: GTR RAxML-NG was called at 24-Jan-2025 19:14:01 as follows: raxml-ng --evaluate --msa raxml/archaea_raxml-check.raxml.reduced.phy --tree ar53_r220_fixed.tree --prefix raxml/archaea_raxml --model GTR+G --threads 24 Base frequencies: 0.220090 0.249290 0.290844 0.239776 Inference[0]: Time 16.044 CAT-based likelihood -0000, best rearrangement setting 5 alpha[0]: 1.000000 rates[0] ac ag at cg ct gt: 1.222614 4.378139 1.877652 0.999680 5.988560 1.000000 NOT conducting any final model optimizations on all 1 trees under CAT-based model .... Final GAMMA likelihood: -282040.011309 ``` -------------------------------- ### Install PICRUSt2 Source: https://github.com/picrust/picrust2/wiki/Manually-install-QIIME-2-plugin Install the latest version of PICRUSt2 using conda. Ensure you are in the correct QIIME 2 environment. ```bash conda install -c bioconda -c conda-forge picrust2 ``` -------------------------------- ### Install PICRUSt2 from Source Source: https://github.com/picrust/picrust2/wiki/Installation Creates and activates a conda environment using the 'picrust2-env.yaml' file, then installs PICRUSt2 in editable mode using pip. This method is suitable for development or when specific source modifications are needed. ```bash mamba env create -f picrust2-env.yaml mamba activate picrust2 pip install --editable . ``` -------------------------------- ### Install PICRUSt2 with Mamba Source: https://github.com/picrust/picrust2/wiki/Quickstart Use mamba to create a dedicated environment for PICRUSt2 and then activate it. This ensures dependency management. ```bash mamba create -n picrust2 -c bioconda -c conda-forge picrust2=2.6.1 mamba activate picrust2 ``` -------------------------------- ### Navigate to placement_tools Directory Source: https://github.com/picrust/picrust2/wiki/Pre-requisite-installations Change the current directory to 'placement_tools' where the pipeline source code is located. This is a prerequisite for installing the tools. ```bash cd placement_tools ``` -------------------------------- ### List Tutorial Files Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(San-Juan,-April-2019) Lists the downloaded tutorial files to verify their presence. ```bash ls ``` -------------------------------- ### Download PICRUSt2 Tutorial Files Source: https://github.com/picrust/picrust2/wiki/q2-picrust2-Tutorial Use these commands to download the necessary biom table, sequences, and metadata files for the tutorial. Ensure you are in the desired directory before running. ```bash mkdir q2-picrust2_tutorial cd q2-picrust2_tutorial wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_biom.qza wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_seqs.qza wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_metadata.tsv ``` -------------------------------- ### Install PICRUSt2 with Conda Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.3-beta) Installs PICRUSt2 version 2.1.3_b and its dependencies into a new Conda environment named 'picrust2'. Ensure you have Miniconda installed. ```bash conda create -n picrust2 -c bioconda -c conda-forge picrust2=2.1.3_b ``` -------------------------------- ### Download Tutorial Files Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(San-Juan,-April-2019) Downloads necessary data files for the PICRUSt2 tutorial. ```bash wget http://kronos.pharmacology.dal.ca/public_files/tutorial_datasets/picrust2_tutorial_files/seqs.fna wget http://kronos.pharmacology.dal.ca/public_files/tutorial_datasets/picrust2_tutorial_files/table.biom wget http://kronos.pharmacology.dal.ca/public_files/tutorial_datasets/picrust2_tutorial_files/metadata.tsv ``` -------------------------------- ### Download PICRUSt2 tutorial test files Source: https://github.com/picrust/picrust2/wiki/Swapping-in-SEPP-for-read-placement-with-q2-picrust2 Download the necessary test files for the PICRUSt2 tutorial, including the ASV count table, ASV sequences, and metadata, into a new directory. ```bash mkdir q2-picrust2_test cd q2-picrust2_test wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_biom.qza wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_seqs.qza wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/mammal_metadata.tsv ``` -------------------------------- ### List Tutorial Files Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.4-beta) Lists the files present in the current directory, which should include 'metadata.tsv', 'seqs.fna', and 'table.biom'. ```bash ls -1 ``` -------------------------------- ### Install STAMP using Conda Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.4-beta) Install the STAMP visualization tool using Conda on Mac or Linux systems. Ensure your current Conda environment is deactivated before installation. ```bash conda deactivate conda install -c bioconda stamp ``` -------------------------------- ### View q2-picrust2 Full Pipeline Help Source: https://github.com/picrust/picrust2/wiki/q2-picrust2-Tutorial Run this command to see the available options and required inputs for the full PICRUSt2 pipeline within QIIME 2. The inputs --i-table and --i-seq are mandatory. ```bash qiime picrust2 full-pipeline --help ``` -------------------------------- ### Install PICRUSt2 with Conda Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.4.2) Installs PICRUSt2 version 2.4.2 and its dependencies into a new Conda environment named 'picrust2'. ```bash conda create -n picrust2 -c bioconda -c conda-forge picrust2=2.4.2 ``` -------------------------------- ### Download and Unzip Tutorial Data Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.4-beta) Downloads the 'chemerin_16S.zip' tutorial data archive, unzips it, and changes the current directory to the extracted 'chemerin_16S' folder. ```bash wget http://kronos.pharmacology.dal.ca/public_files/picrust/picrust2_tutorial_files/chemerin_16S.zip unzip chemerin_16S.zip cd chemerin_16S ``` -------------------------------- ### Install PICRUSt2 with Conda Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.4-beta) Installs PICRUSt2 version 2.1.4_b and its dependencies into a new Conda environment named 'picrust2'. ```bash conda create -n picrust2 -c bioconda -c conda-forge picrust2=2.1.4_b ``` -------------------------------- ### Create and Enter Output Directory Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Streamlined-Tutorial-(v2.1.4-beta) Before running the PICRUSt2 pipeline, create a dedicated directory for output files and navigate into it. ```bash mkdir picrust2_out_pipeline cd picrust2_out_pipeline ``` -------------------------------- ### Install eggNOG-mapper and Download Databases Source: https://github.com/picrust/picrust2/wiki/Creating-the-PICRUSt2-database Installs eggNOG-mapper and downloads necessary databases for genome annotation. This is a prerequisite for running eggNOG on reference genomes. ```bash #conda create -n eggnog #conda activate eggnog #conda install -c bioconda -c conda-forge eggnog-mapper #conda doesn't work well with compute canada #having issues unzipping things on compute canada. Downloading to vulcan and then copying folder across #wget https://github.com/eggnogdb/eggnog-mapper/archive/refs/tags/2.1.12.zip #unzip 2.1.12.zip #scp -r eggnog-mapper-2.1.12/ rwright@graham.alliancecan.ca:/home/rwright/scratch/ cp -r scratch/eggnog-mapper-2.1.12/ tools/ #been having issues with not much storage space available - asked Ben to move his things python setup.py install cd .. cd .. cd scratch mkdir eggnog_data download_eggnog_data.py --data_dir eggnog_data create_dbs.py -m diamond --dbname bacteria --taxa Bacteria --data_dir eggnog_data create_dbs.py -m diamond --dbname archaea --taxa Archaea --data_dir eggnog_data ``` -------------------------------- ### Get help for qiime picrust2 custom-tree-pipeline Source: https://github.com/picrust/picrust2/wiki/Swapping-in-SEPP-for-read-placement-with-q2-picrust2 Run this command to view the help documentation for the qiime picrust2 custom-tree-pipeline command, which outlines its required inputs and available options. ```bash qiime picrust2 custom-tree-pipeline --help ``` -------------------------------- ### Example Output of marker_predicted_and_nsti.tsv Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.1.3-beta) This is an example of the output format for the `marker_predicted_and_nsti.tsv` file, showing ASV identifiers, 16S rRNA gene counts, and NSTI scores. ```text sequence 16S_rRNA_Count metadata_NSTI 20e568023c10eaac834f1c110aacea18 1 0.008994 23fe12a325dfefcdb23447f43b6b896e 1 0.047617 288c8176059111c4c7fdfb0cd5afce64 4 0.44977799999999996 2a45f0b7f68a35504402988bcb8eb7fe 1 0.000101 343635a5abc8d3b1dbd2b305eb0efe32 4 0.5681 ... ``` -------------------------------- ### Example Stratified Metagenome Contribution Table Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.3.0-beta) An example of the stratified metagenome contribution table, showing how individual ASVs contribute to gene family abundances within each sample. ```text sample function taxon taxon_abun taxon_rel_abun genome_function_count taxon_function_abun taxon_rel_function_abun 100CHE6KO EC:1.1.1.1 20e568023c10eaac834f1c110aacea18 26.0 1.9607843137254901 2 52.0 3.9215686274509802 100CHE6KO EC:1.1.1.1 288c8176059111c4c7fdfb0cd5afce64 25.5 1.9230769230769231 1 25.5 1.9230769230769231 100CHE6KO EC:1.1.1.1 343635a5abc8d3b1dbd2b305eb0efe32 16.0 1.206636500754148 1 16.0 1.206636500754148 ``` -------------------------------- ### Run Full PICRUSt2 Pipeline (Old IMG Database) Source: https://github.com/picrust/picrust2/wiki/Workflow Executes the entire PICRUSt2 pipeline using the previous PICRUSt2-oldIMG database. This command replicates the behavior of earlier versions. ```bash picrust2_pipeline_singleRef.py -s study_seqs.fna -i study_seqs.biom -o picrust2_out_pipeline_split -p 1 ``` -------------------------------- ### Example Unstratified Metagenome Prediction Table Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.3.0-beta) An example of the unstratified metagenome prediction table format, where rows represent gene families (functions) and columns represent samples. ```text function 100CHE6KO 101CHE6WT 102CHE6WT ... EC:1.1.1.1 1432.0 1967.5 1408.5 ... EC:1.1.1.100 2034.0 2748.16 2443.5 ... EC:1.1.1.103 0.0 0.0 0.0 ... ``` -------------------------------- ### Example Output of EC_predicted.tsv Source: https://github.com/picrust/picrust2/wiki/PICRUSt2-Tutorial-(v2.1.3-beta) This is an example of the output format for the `EC_predicted.tsv` file, showing ASV identifiers and predicted copy numbers for various Enzyme Classification (EC) numbers. ```text sequence EC:1.1.1.1 EC:1.1.1.10 EC:1.1.1.100 ... 20e568023c10eaac834f1c110aacea18 2 0 3 ... 23fe12a325dfefcdb23447f43b6b896e 0 0 1 ... 288c8176059111c4c7fdfb0cd5afce64 1 0 1 ... ... ```