Apr 30, 2024

Public workspaceFUNDIS ONT Sequencing IT/Compute Pop!_OS 22.04 Setup

 Forked from ONT Sequencing IT/Compute Pop!_OS 22.04 Setup
This protocol is a draft, published without a DOI.
FUNDIS ONT Sequencing IT/Compute Pop!_OS 22.04 Setup
  • 1Mycota Lab;
  • 2The Hoosier Mushroom Society;
  • 3FUNDIS
Harte Singer
Open access
Protocol Citation: Stephen Douglas Russell, Harte Singer 2024. FUNDIS ONT Sequencing IT/Compute Pop!_OS 22.04 Setup. protocols.io https://protocols.io/view/fundis-ont-sequencing-it-compute-pop-os-22-04-setu-dcwy2xfw
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 06, 2024
Last Modified: April 30, 2024
Protocol Integer ID: 99000
Keywords: Oxford Nanopore Technologies, DNA sequencing, DNA barcoding, MinION, Flongle, system76, linux, POP! OS
Abstract
These are the steps to setting up a Linux system running Ubuntu for ONT Nanopore sequencing, basecalling, and consensus-building of fungal amplicon sequences. Adapted from dx.doi.org/10.17504/protocols.io.14egn7kzmv5d/v3

Includes an R-based run QC script
Preparing a new CPU for MinION Sequencing
Preparing a new CPU for MinION Sequencing
Ideally you will be running a linux machine with a Ubuntu distro with an NVIDIA graphics card with at least 8GB of ram. (I am running a 4070Ti) You should have at least 32GB of system ram and a new processor.



Minimum IT requirements for MinION from ONT: Download minion-it-reqs.pdfminion-it-reqs.pdf


Install CUDA toolkit - https://developer.nvidia.com/cuda-toolkit :
Command
wget https://developer.download.nvidia.com/compute/cuda/repos/ubuntu2204/x86_64/cuda-ubuntu2204.pin
sudo mv cuda-ubuntu2204.pin /etc/apt/preferences.d/cuda-repository-pin-600
wget https://developer.download.nvidia.com/compute/cuda/11.7.0/local_installers/cuda-repo-ubuntu2204-11-7-local_11.7.0-515.43.04-1_amd64.deb
sudo dpkg -i cuda-repo-ubuntu2204-11-7-local_11.7.0-515.43.04-1_amd64.deb
sudo cp /var/cuda-repo-ubuntu2204-11-7-local/cuda-*-keyring.gpg /usr/share/keyrings/
sudo apt-get update
sudo apt-get -y install cuda

Install Boost

Command
sudo apt install libboost-all-dev

Install MinKNOW from https://community.nanoporetech.com/downloads?from=support

Specifically download the GPU version of the software with the following name. It is a .deb file and your computer will install it when you double-click it

MinKNOW Software for the MinION Mk1B and the PromethION 2 Solo (P2 Solo) Devices

Install Guppy from the "Archived Software" section at the bottom of this page https://community.nanoporetech.com/downloads?from=support

Guppy v6.5.7

Make sure you install the Ubuntu 20 GPU version, just double click the installer and it should work.








Per this document: https://denbi-nanopore-training-course.readthedocs.io/en/latest/read_qc/MinionQC.html
Install R: https://cran.r-project.org/
Install MinionQC: https://github.com/roblanf/minion_qc

Install R:

Command
# update indices
sudo apt update -qq
# install two helper packages we need
sudo apt install --no-install-recommends software-properties-common dirmngr
# add the signing key (by Michael Rutter) for these repos
# To verify key, run gpg --show-keys /etc/apt/trusted.gpg.d/cran_ubuntu_key.asc 
# Fingerprint: E298A3A825C0D65DFD57CBB651716619E084DAB9
wget -qO- https://cloud.r-project.org/bin/linux/ubuntu/marutter_pubkey.asc | sudo tee -a /etc/apt/trusted.gpg.d/cran_ubuntu_key.asc
# add the R 4.0 repo from CRAN -- adjust 'focal' to 'groovy' or 'bionic' as needed
sudo add-apt-repository "deb https://cloud.r-project.org/bin/linux/ubuntu $(lsb_release -cs)-cran40/"


Command
sudo apt install --no-install-recommends r-base
Open an R terminal window for the following command
Command
install.packages(c("data.table", 
                   "futile.logger",
                   "ggplot2",
                   "optparse",
                   "plyr",
                   "readr",
                   "reshape2",
                   "scales",
                   "viridis",
                   "yaml"))

Install Bioconductor:

In an R command window:

Command
if (!require("BiocManager", quietly = TRUE))
    install.packages("BiocManager")
BiocManager::install(version = "3.15")

Install Anaconda:
from: https://www.digitalocean.com/community/tutorials/how-to-install-the-anaconda-python-distribution-on-ubuntu-22-04


Command
cd /tmp

Command
curl https://repo.anaconda.com/archive/Anaconda3-2022.05-Linux-x86_64.sh --output anaconda.sh

Command
You can now verify the data integrity of the installer with cryptographic hash verification through the SHA-256 checksum. You’ll use the sha256sum command along with the filename of the script:
sha256sum anaconda.sh

Expected result
You’ll receive output that looks similar to this:
fedf9e340039557f7b5e8a8a86affa9d299f5e9820144bd7b92ae9f7ee08ac60 anaconda.sh


Command
bash anaconda.sh

Expected result
Press ENTER/yes as needed
Welcome to Anaconda3 2021.11

In order to continue the installation process, please review the license
agreement.
Please, press ENTER to continue
>>>


Command
source ~/.bashrc

Command
conda list

Expected result


# packages in environment at /home/user/anaconda3:
#
# Name Version Build Channel
_ipyw_jlab_nb_ext_conf 0.1.0 py39h06a4308_0
_libgcc_mutex 0.1 main
_openmp_mutex 4.5 1_gnu
alabaster 0.7.12 pyhd3eb1b0_0
anaconda 2022.05 py39_0



Command
conda search "^python$"



Command
Verify Python is installed
python --version


Command
Install bioconda
conda install -c bioconda seqkit



Install NGSpeciesID: https://github.com/ksahlin/NGSpeciesID


Command
conda create -n NGSpeciesID python=3.6 pip 
conda activate NGSpeciesID

Command
conda install --yes -c conda-forge -c bioconda medaka==0.11.5 openblas==0.3.3 spoa racon minimap2
pip install NGSpeciesID

Command
conda activate NGSpeciesID

Command
Test the install
mkdir test_ngspeciesID
cd test_ngspeciesID

Command
Download the test fastq file called "sample_h1.fastq" (filesize 390kb)
curl -LO https://raw.githubusercontent.com/ksahlin/NGSpeciesID/master/test/sample_h1.fastq

Command
Run the NGSpecies command on test file. Outputs will be saved in "/test_ngspeciesID/sample_h1/", where the final polished consensus file ("consensus.fasta") is located in the "/test_ngspeciesID/sample_h1/medaka_cl_id_" directory.
NGSpeciesID --ont --fastq sample_h1.fastq --outfolder ./sample_h1 --consensus --medaka

You should now be ready to begin sequencing runs.
Installing Dorado
Installing Dorado
We will eventually be moving to Dorado basecalling.

Installing Dorado:

sudo -i

sudo apt-get update && apt-get install -y --no-install-recommends \
curl \
git \
ca-certificates \
build-essential \
nvidia-cuda-toolkit \
libhdf5-dev \
libssl-dev \
libzstd-dev \
cmake \
autoconf \
automake
git clone https://github.com/nanoporetech/dorado.git dorado
cd dorado
cmake -S . -B cmake-build
cmake --build cmake-build --config Release -j
ctest --test-dir cmake-build



pip install pre-commit
pre-commit install