Readme For runningt4f.pl, KO2Path and Path2Class

These Scripts Are developed to help researchers analyze Tax4Fun Output and carry out a in-depth functional analysis of the Amplicon Metagenomics dataset. These scripts will take Tax4Fun Output and fetch all the KO IDs from them and then connect to Kegg Server to extract:

1. KO2Path: Pathway Information for each KO ID.
2. Path2Class: Class and Subclass of each Pathway identified in previous step.

Each Step creates output as a normalized profile matrix having respective information in coloumns, where each coloumn denotes the sample ID.

The entries from KEGG are saved on Your local disk so that we dont have to connect to internet to retrive the files everytime we would like to run the script.

Both KO2Path and PathClass are provided with function of inputting keyword, to analyze context specific pathway or class:

1. If provided in KO2Path there will be an additional pathway keyword profile generated, with all the molecules whose pathways have the keyword in thier name. e.g. if you would like to explore all
   molecules that are from pathway like Methane metabolism present in
   your dataset then in KO2Path step you can use key word
   "Methane metabolism".
2. If provided in Path2Class there will be an additional class keyword profile generated, with all the pathways whose class/sub-class title has your keyword. e.g. if you would like to explore all
   pathways that are from a perticular sub-class like Energy metabolism present in your dataset then in Path2Class step you can use key word
   "Energy".

Pre-requisites: cURL- (usually installed on linux) available from most Linux Repositories or source from : https://curl.haxx.se/download.html

Tax4Fun-To run and acquire output. Tax4Fun can be downloaded from : http://tax4fun.gobics.de/
Tax4Fun has its own requirements that includes R-statistical package Tax4Fun SILVA reference data (available on the link above)

PERL- ALso Installed on LInux by default if not would be available On repository, if still not download from: https://www.perl.org/get.html#unix_like

Statistics::R package: install with the command: sudo cpan install Statistics::R

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Please NOTE: At every stage when requested to enter the path make sure you enter the path with the trailing "/";

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Please cite: Mehetre, G., Shah, M., Dastager, S.G. et al. Arch Microbiol (2018). https://doi.org/10.1007/s00203-018-1484-4

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STEP1: Run Tax4Fun

Note the path of tsv format of Qiime Biom made using SILVA. if biom is in binary/hdf5 format convert it using: biom convert -i <BIOM FILE> -o <OUT FILE NAME> --to-tsv --header-key taxonomy

You can run Tax4Fun with the Script provided: perl runningt4f.pl first you will be prompted for SILVA reference data Folder, usually ending with : "SILVA123/" for version 123. please enter full path.

Next you will be prompted for FOLDER where the input file and output file would be present. (please note: do not provide path to input file here)

Then you will be prompted for input tsv file name which would be present in the above folder

STEP2: Run KO2Path

In this step we will convert the Tax4Fun Output into a Pathways profile based on information present on KEGG database and Tax4Fun output.

Make sure your internet connection is working. If you are behind a institution proxy you will need to enter the following command: export http_proxy=http://<proxyUsername>:<proxyPassword>@<Proxyaddress>:<Port>

KO2Path usage : ./KO2Path "" <KEGG_Storage_folder>

This has to be run from the folder where the script is present

would have the COMPLETE path of the Tax4fun output.

would be the pathway whose all the molecule you would like to see specially in a seperate file. Leave blank if not required but still leave "".

<KEGG_Storage_folder> would be the folder where you would like to store the KEGG files for quicker access. make sure the directory exists.

The output would be saved in the same folder as your input file.

STEP3: Run Path2Class

In this step we will convert the KO2Path Output into a Class/Sub-class profile based on information present on KEGG database and Tax4Fun output.

KO2Path usage : ./Path2Class "" <KEGG_Storage_folder>

This has to be run from the folder where the script is present

would have the COMPLETE path of the KO2Path output.

would be the pathway whose all the molecule you would like to see specially in a seperate file. Leave blank if not required but still leave "".

<KEGG_Storage_folder> would be the folder where you would like to store the KEGG files for quicker access. make sure the directory exists.

The output would be saved in the same folder as your input file.