ONTvariants - QC

Create working folder and copy data

Let’s create an interactive session on the HPC:

Let’s create working folders for today. Copy and paste the following block of code into your terminal and hit enter for the last command:

Now, let’s copy the scripts and data for today’s session:

Install tools using conda

Approach 1: Create a conda environment and install tools one at a time

Create a conda environment called ONTvariants_QC

Let’s activate the conda environment:

Next, we need to install few tools for today’s exercises. Now let’s go the https://anaconda.org and search for the following tools and instructions on how to install them:

For example, search for nanoplot:

Open

If the tool you are looking is available in conda, a list of options will be presented. Typically choose the option at the top with most downloads and compatible for your system:

Open

Click on the link to the tool of interest and you will be presented with the conda command line to run in your system to install the tool:

Copy and paste the first command shown above in your terminal where you have activated the ‘ONTvariant’ conda environment (Approx. 5-10min):

Now repeat the process for porechop_abi (Approx. 1min)’, then install it (Approx. 1min):

Next, let’s install chopper (Approx. 1min):

Finally, let’s install the seqkit suite of tools:

Approach 2: Create environment and install tools all at once

This is a slower option, but it is convenient when installing many tools.

Prepare the following environment.yml file:

Create a new environment:

Running QC

Now that we have installed all the tools needed for the QC of Nanopore reads, let’s run the preprocessing of reads.

Let’s initially move to the run1_QC working directory:

Now let’s copy the script for the exercise:

Note: the above script copies the launch script for the scripts folder to the current directory denoted by the full stop “ . “ at the end of the command.

Let’s print the content of the script:

Note:

• Line 1: Defines that the script is a bash script.

• Lines 2-5: Are commented out with “#” at the beginning and are ignored by bash, however, these PBS lines tell the scholar (PBS Pro) the name of the job (line 2), the number of CPUs and RAM memory to use (line 3), the time to run the script (line 4) and report if there are any errors (line 5).

• Line 7: Tells the job to run on the current directory.

• Line 9: Activate the conda environment where the QC tools were installed using conda.

• Lines 11-17: User defined variables. Modify the FASTQ, genome and/or sample ID to use to run the job as appropriate. Note: in the lines below, the variable names are used instead of the actual names or locations of the files (e.g., $FASTQ)

• Line 20: Run a Quality Control (QC) overview of the raw Nanopore reads using NanoPlot

• Line 23: Remove adapter sequences from the 5'- and 3’-ends of the raw reads

• Line 26: Filter reads with a quality score below Q10 (90% accuracy; -q 10) and shorter than 300 bases (-l 300)

• Line 28: collect the stats for trimmed FASTQ files processed using porechop_abi and chopper

Submit the QC job to the HPC cluster:

Monitor the progress of the job:

The job will take ~30 - 40 min to complete.

Outputs

The following list of outputs will be generated once the job has completed:

As outputs find the porechop_abi processed file (SRR17138639_1_porechop_abi.fastq) and the chopper output (SRR17138639_1_porechop_abi_chopper_q10_300b.fastq). To visualise the QC reports, let’s connect to the HPC via file finder (see below).

NOTE: To proceed, you need to be on QUT’s WiFi network or signed via VPN.

To browse the working folder in the HPC type in the file finder:

Windows PC

Mac

Once connected, browse to the “/ONTvariants/runs/run1_QC” folder.

Let’s open the “SRR17138639_QC_NanoPlot-report.html“ report:

Summary statistics

Next, let’s inspect the “SRR17138639_QC_LengthvsQualityScatterPlot_dot.png“ file. Alternatively for high resolution image open instead “SRR17138639_QC_LengthvsQualityScatterPlot_dot.html“

Next: ONTvariants - mapping