TITLE: ONT Sequencing IT/Compute Pop!_OS 22.04 Setup
AUTHORS: Stephen Douglas Russell

[DESCRIPTION]
The IT requirements for processing MinION data should be carefully reviewed before purchasing a MinION device.  You will want to go with a Linux system. System76 is really the primary/best vendor for laptops. Pay careful attention to the GPU. It is probably the most important component for fast processing of the data. Here is is a link to a Facebook thread of some discussion when first considering the specs required. 

Setting up all of the programs/dependencies, particularly for utilizing the GPU during the analytical workflows is the next important step. You will want to get all of this in place before you start with the lab workflows, as there are many things that could go wrong or that you will need to work through in order for you to be able to actually begin a run.

[STEPS]
SECTION: Preparing a new CPU for MinION Sequencing
1. The final setup I went with can be found below. It was expensive (around   for the laptop in early 2022), but should be able to achieve live basecalling for two MinION devices at the same time. Overall specs of my laptop: 

 
 

Minimum IT requirements for MinION from ONT:    
SECTION: Preparing a new CPU for MinION Sequencing
2. The remainder of this protocol assumes you have completed all of the preliminary setup steps that are common with any new CPU. 

Install CUDA toolkit - https://developer.nvidia.com/cuda-toolkit :
 

SECTION: Preparing a new CPU for MinION Sequencing
3. Install Boost

 

SECTION: Preparing a new CPU for MinION Sequencing
4. The process at this link was instrumental to this protocol. It is recreated and simplified here. ORIGINAL PROTOCOL. It was written for Pop!_OS 21.04. The following protocols also work with Pop!_OS 22.04. I would follow the steps at the link rather than here so you get a broader context of the actions you are performing on your system.


 

 

 


 





SECTION: Preparing a new CPU for MinION Sequencing
5. Add the Focal repos:
 
Copy and paste the following into your file:


X-Repolib-Name: Pop_OS System Sources Enabled: yes Types: deb deb-src URIs: http://us.archive.ubuntu.com/ubuntu/ Suites: focal focal-security focal-updates focal-backports Components: main restricted universe multiverse X-Repolib-Default-Mirror: http://us.archive.ubuntu.com/ubuntu/


 

 

SECTION: Preparing a new CPU for MinION Sequencing
6. Pin the Focal repos. Start by creating another new file with nano:


 

Copy and paste the following into your file:


Package: * Pin: release n=focal* Pin-Priority: 10

Check that is was created correctly


 

 


 

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7. Install MinKNOW and required packages


 

SECTION: Preparing a new CPU for MinION Sequencing
8. Install ONT Guppy
 

 

 

 

 

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9. Setup the MinKnow service

 

 

 

 
Copy the following to your new file:


[Unit] Description=Service to manage the guppy basecall server. Documentation=https://community.nanoporetech.com/protocols/Guppy-protocol/v/GPB_2003_v1_revQ_14Dec2018  [Service] Type=simple ExecStart=/opt/ont/guppy/bin/guppy_basecall_server --log_path /var/log/guppy --config dna_r9.4.1_450bps_fast.cfg --port 5555 -x cuda:all  Restart=always User=root MemoryLimit=8G MemoryHigh=8G CPUQuota=200%  [Install] Alias=guppyd.service WantedBy=multi-user.target

Check the file:


 

 

 

MinKNOW GUI should now be available in your programs. Validate that it opens correctly.
SECTION: Preparing a new CPU for MinION Sequencing
10. Changes to MinKnow file permissions at the bottom here:
https://gringer.gitlab.io/presentation-notes/2021/10/08/gpu-calling-in-minknow/

"For my computer, there’s an issue with MinKNOW not being able to access or create files. As a “nuclear” option, Miles Benton suggested changing the user and group for the minknow service to root"

 

SECTION: Preparing a new CPU for MinION Sequencing
11. 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:

 


 

 

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12. Install Bioconductor:

In an R command window:

 

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


 

 

 

 

 

 

 

 

 

 

 

 

 

 

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


 

 

 

 

 

 

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15. You should now be ready to begin sequencing runs.
SECTION: Updating MinKnow &amp; Installing Dorado
17. Since this protocol was first written, MinKnow needed an update for the 10.4.1 Flongle cells and then again to bring in Dorado basecaclling. The steps to perform the update can be found below:

 

These dependency versions will likely keep changing with time.
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16. 
SECTION: Updating MinKnow &amp; Installing Dorado
18. Installing Dorado: