LMToolkit is a small command line pipeline to simplify going from ONT raw data to basecalling using Guppy to Assembly using Flye and Alignments using Guppy_Aligner, all of the tools are existing tools. This pipeline is designed to be beginner friendly for people new to Nanopore Bioinformatics and Bioinformatics as a whole. The input command used is shown for each program to help guide advanced usage of the linked programs. It is constantly being updated and tweaked for new nanopore updates and to enhance the user experience. The install file contains the basic commands to get started. Please make an issue thread if you have any questions!
This pipeline is only for Linux due to its reliance on Flye and the GPU Based Version of ONT's Guppy. It may run on windows using WSL2 and Ubuntu. (The setup is for debian based Linux Distros due to APT but should run on other Linux distros if Flye and Python3 are installed manually)
GETTING STARTED
To get started you will
cd to the LMToolkit directory
(Install commands located in INSTALL file)
run LMToolKit_Setup.py (python3 LMToolKit_Setup.py) this will run "sudo apt-get update" , "sudo apt-get install python3.8" and "conda install flye". These will install python3.8 and flye for you if you do not already have them.
The program next will create a directory in Documents; Ie Documents/LMToolkit/ with /MinionOut and /Assembly_Out as subdirectories.
It will then ask for the location of your guppy_basecaller and guppy_basecall config file 450_bps_hac.cfg is only recommended on high end PCs 450_bps_fast.cfg is recommended for most read the readme in ont-guppy for more info!
If you want to change the config file just rerun the setup.
RUNNING LM_TOOLKIT:
Now that the setup is done cd to LM_Toolkit and run LMToolkit.py in terminal. (python3 LMToolkit.py)
The program is going to ask for the location of the fast5 folder created by Minknow, what you wish to name the output folder and whether your sample contained plasmids (used by Flye for assembly).
The outputs from Guppy will be found in /MinionOut as .fastq files the program will also combine them into a singular .fastq file named what you named the output without modifying any of the original FASTQ files.
The output from Flye will be found in /Assembly_Out as assembly.fasta.
All information and errors from the individual program will also show in the terminal along with the programs data as well.
I hope this simplifies analysis for anyone new to ONT basecalling and assembly!
https://github.com/fenderglass/Flye required (installed during setup)
and
ONT's GPU Based Guppy Required (needs to be installed from ONT) version 4.2.2.
if flye does not install during setup it is mostly likely because bioconda is not installed. Try to first install bioconda:
sudo apt update
sudo apt install curl
curl -O https://repo.anaconda.com/miniconda/Miniconda3-latest-Linux-x86_64.sh
sh Miniconda3-latest-Linux-x86_64.sh
conda config --add channels defaults
conda config --add channels bioconda
conda config --add channels conda-forge