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Lab 7 NOTES - GGG 201b, Feb 24, 2023

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Friday Lab links

Logging in

:::spoiler Logging into farm and running RStudio Server

Quick start: logging into farm and running RStudio Server

Log into farm with ssh/MobaXterm - link.

Then run:

srun -p high2 --time=3:00:00 --nodes=1 \
    --cpus-per-task 4 --mem 10GB --pty /bin/bash

and wait for a new prompt. Then run:

module load spack/R/4.1.1
module load rstudio-server/2022.07.1
rserver-farm

Then,

  • in a new shell, run the ssh tunneling command output by rserver-farm above;
  • connect to the localhost URL output by rserver-farm;
  • Login with your username and the one-time password output by rserver-farm;
  • start a Terminal. :::

Install assembly software

Create a new conda/mamba environment like so:

mamba create -n assembly -y megahit prokka quast

Question: for those of you familiar with the modules subsystem, what modules would you want to load here?

Activate software environment & change to lab7 working directory

conda activate assembly
mkdir -p ~/lab7/
cd ~/lab7/

Copy in some raw data files

Let's copy in some paired-end sequencing from the E. coli Long Term Evolution Experiment:

cp ~ctbrown/data/ggg201b/SRR2584857_*.fastq.gz .

This gives us two files, containing paired end sequencing:

$ ls -lh

>total 180M
>-rw-rw-r-- 1 datalab-02 datalab-02 180M Feb 24 >07:41 SRR2584857_1.fastq.gz
>-rw-rw-r-- 1 datalab-02 datalab-02 180M Feb 24 07:41 SRR2584857_2.fastq.gz

Generate an assembly

MEGAHIT is an assembler for microbial genomes and metagenomes that is fast and memory efficient; it's my first suggestion for looking at data, even though you might end up getting better results with a different assembler in the end.

Run the megahit assembler:

megahit -1 SRR2584857_1.fastq.gz -2 SRR2584857_2.fastq.gz -f -m 5e9 -t 4 -o SRR2584857_assembly

and check out the contents of the assembly file:

head SRR2584857_assembly/final.contigs.fa

Save the file into a nicer name:

cp SRR2584857_assembly/final.contigs.fa SRR2584857-assembly.fa

Summarize the assembly

The quast software is a nice way to generate some summary statistics.

Run quast to summarize the assembly:

quast SRR2584857-assembly.fa

and look at the results:

cat quast_results/latest/report.txt

Annotate the assembly using prokka:

The prokka software does a really nice job of annotating bacterial genomes!

prokka --prefix SRR2584857_annot SRR2584857-assembly.fa

We will use this set of annotations and information for the next step - viewing the genome assembly!

Using IGV

GGG201B Lab: Visual exploration of genomic data with the Integrative Genomics Viewer (IGV)