MTN Region SARS-CoV-2 Training

By: Joel Sevinsky

June 26, 2020

Training Prerequisites

In order for this training to be effective for all students participating, each student should perform the following before the first class:

Confirm that they can connect to their VM.
Watch a 75 minute video on the linux operating system.
Watch a short video on tmux, a linux application that allows for multiple terminals.

Connect to your VM

In the slack channel will be listed your VM IP address, along with a ssh key file. This information will be posted on Monday, June 29th. In order to connect to this VM, you will need to download the terminal program MobaXterm. MobaXterm can be downloaded from https://mobaxterm.mobatek.net/download-home-edition.html. IMPORTANT: Please download the “Portable edition”, not the “Installer edition”. The “Portable edition” will allow you to run the application without installing it, thus no admin rights needed. Once you have downloaded MobaXterm and have access to your IP address and ssh key, watch the youtube video on How to Connect to Your VM. I will also post the username and passphrase in slack.

Linux video

The video link is https://www.youtube.com/watch?v=oxuRxtrO2Ag. This video will give you a basic understanding of navigating around the bash terminal in linux. It is well worth the 75 minutes before the course, working within your own VM.

Tmux video

Many of the processes we will be running will require long processing times that could be interrupted by network connection issues. To avoid this, we will perform the exercises in the class using the tmux application. This tmux tutorial video will guide you through the process of using tmux.

Some additional resources that you will find useful when starting out are:

Agenda Overview

Session 1 - Linux - Monday July 6th, 12 PM and 2 PM MDT
Session 2 - Background on Viral Genomics and Coronaviruses - Tuesday, July 7th, 12 PM and 2 PM MDT
Session 3 - Sequencing Methods for SARS-CoV-2 - Wednesday July 8th, 12 PM and 2 PM MDT
Session 4 - StaPH-B Toolkit - Monday July 13th, 12 PM and 2 PM MDT
Session 5 - UPHL BioNGS - Tuesday July 14th, 12 PM and 2 PM MDT
Session 6 - Commercial Options - Wednesday July 15th, 12 PM and 2 PM MDT
Session 7 - NGS Data Visualization for QC of Results - Monday July 20th, 12 PM and 2 PM MDT
Session 8 - Data Sharing with GISAID and NCBI - Tuesday July 21st, 12 PM and 2 PM MDT
Session 9 - Data Visualization in Nextstrain - Wednesday July 22nd, 12 PM and 2 PM MDT

Office hours will be offered each week on Thursday from 2 PM to 4 PM MDT, on Friday from 9 AM to 11 AM MDT, and by request.

Expanded Agenda (will contain links, videos, code, as course progresses)

Week 1

Session 1 - Linux

Recording for Session 1 - 12:00 PM MDT

Recording for Session 1 - 3:00 PM MDT

Linux basics
Working in Tmux
- Tmux Cheat Sheet
- Tmux Video
Connecting to Basespace
- Basespace CLI
Connecting to cloud resources
- gsutil
Transferring data to your VM for the class

Commands used in this session

***bash commands***

dir                           #lists out a directory
ls                            #also lists out a directory
ls -la                        #lists out a directroy with details
ll                            #alias for ls -la
cd <directory>                #change directory
cd                            #will bring you to your home directory
mkdir <dir>                   #make directory
rmdir <dir>                   #remove directory if it is empty
rm -rf <dir>                  #remove directory if it is full
gzip <name.fastq>             #zip a read file

***tmux***

tmux ls                       #list open tmux sessions
tmux new -s <name>            #start a tmux session called <name>
tmux a -t <name>              #attach to tmux session called <name>
tmux kill-session -t <name>   #kill tmux session called <name>
Ctrl-b ,                      #rename current window
Ctrl-b c                      #create new window
Ctrl-b n                      #go to next window
tmux info                     #help

***basespace cli***

bs auth                                   #authenticate to Basespace
bs list projects                          #list projects in basespace
bs download project -n <name> -o <dir>    #download project files to <dir>

***gsutils***

gsutil ls                                 #list storage buckets
gsutil cp <source> <destination>          #cp data from storage bucket

***sra-toolkit*** - might need to "sudo apt-get install sra-toolkit"

prefetch <sra_id>
fastq-dump --split-files --gzip <sra_id>

Session 2 - Background on Viral Genomics and Coronaviruses

Recording for Session 2 - 12 PM MDT

Recording for Session 2 - 3 PM MDT

Session 2 Slides

Viral genomics primer
Considerations of bacteria vs viral pathogens
Coronavirus, the new flu.
Papers on Coronaviruses

REMINDER 7/7 –> register for a GISAID account

Session 3 - Sequencing Methods for SARS-CoV-2

Recording for Session 3 - 12 PM MDT

Recording for Session 3 - 3 PM MDT

Metagenomics, enrichment, amplicon
The ARTIC protocol
ARTIC + Illumina DNA Flex at protocols.io.

Week 2

Session 4 - StaPH-B Toolkit

Recording for Session 4 - 12 PM MDT (Watch the 3 PM instead)

Recording for Session 4 - 3 PM MDT (This is the better recording)

Monroe pipeline
pe_assembly
cluster_analysis

Documentation on Monroe

Some commands to make today’s session easier

tmux new -s session4

mkdir session4-1
cd session4-1
gsutil ls
gsutil ls gs://mtn-reads/
gsutil -m cp -r gs://mtn-reads/session4/* .
tar -xf reads.tar
mkdir reads
mv *.gz reads
rm reads.tar
staphb-wf monroe
staphb-wf monroe pe_assembly
staphb-wf monroe pe_assembly --primers V3 --output pe_assembly_1 --config 20-07-11_pe_assembly.config reads

staphb-wf monroe cluster_analysis
staphb-wf monroe cluster_analysis --output cluster_analysis_1 --config 20-07-11_cluster_analysis.config pe_assembly_1/assemblies/

Session 5 - UPHL BioNGS

Cecret SARS-CoV-2 pipeline

Recording for Session 5 - 12 PM MDT

Recording for Session 5 - 3 PM MDT

Data files

session5_files.tar

session5_files_2.tar

Some commands to make today’s exercises easier

#see if you have a tmux session open
tmux ls

#create a new tmux session called session5
tmux new -s session5

#create new window in your tmux session
<create new window in tmux with Ctrl-b, c>

#copy over session files
gsutil -m cp -r gs://mtn-reads/session5/* .

#extract the session files
tar -xf session5_files_2.tar

#change dir into cecret_session5
cd cecret_session5

#check out covid_samples.txt
column -t covid_samples.txt | less -S

#copy new Cecret.nf to your Cecret folder
cp Cecret.nf ~/Cecret/

#check thatt Cecret.nf was updated
ll ~/Cecret

#launch Cecret - will take about 50 minutes with 10 specimens
~/nextflow run ~/Cecret/Cecret.nf -c cecret.docker.nextflow.google.config

#go up one directory to session5
cd ..

#unpack bakeshow data
tar -xf session5_files.tar

#move into bakeshow directory
cd cecret_bakeshow

#check out run_results.txt
column -t run_results.txt | less -S

Session 6 - Cecret and Monroe Continued

UPDATE: Most likely we will continue looking at Monroe and Cecret results

Recording for Session 6 - 12 PM MDT

Recording for Session 6 - 3 PM MDT

Not covered: