Transcriptome assembly workflow
Workflow for transcriptome assemblies in the Griffith University High Performance Computing (HPC) Cluster (Gowonda)
Based on the workflow from Carmel McDougall, with modifications by Tessa M. Page
License: CC BY-NC-SA 4.0
First-time Gowonda/HPC users (or for those that need a detailed refresher) start with the Beginners guide to Gowonda HPC.
1) Run a quality control with FastQC
Sequenced reads are normally provided in FASTQ format, which is a text-based format similar to FASTA but it also includes quality scores. Transfer the compressed FASTQ files into Gowonda (see section 3 of the “Beginners guide to Gowonda HPC” if you don’ know how to do this!).
After you’ve transferred your FASTQ files into Gowonda, I recommend moving it into a new folder in your scratch, for example transcriptome_raw.
Now you need to run a scheduler script for FastQC, below is an example. Before running the script, I recommend creating a folder called fastqc for your converted output files.
#!/bin/bash -l
#PBS -m abe
#PBS -M your_email_adress@griffith/griffithuni.edu.au
#PBS -N fastqc
#PBS -l select=1:ncpus=1:mem=10gb,walltime=04:00:00
cd $PBS_O_WORKDIR
module load anaconda3/2021.11
source activate labmcdougallRoot
fastqc --noextract -o fastqc/ RNA_1.fastq.gz
fastqc --noextract -o fastqc/ RNA_2.fastq.gz
Download the the summarised FastQC reports (the HTML output files) in the fastqc folder to your computer and open in a web browser to check all the quality information. If you want more details and exact values for the different quality tests, check the output ZIP files.
For guides on how to interpret the FastQC outputs check Babraham Bioinformatics (the developer) and/or this FastQC tutorial.
2) Assembly with Trinity
If paired-end reads were generated, they need to be assembled into transcripts, which Trinity does!
I recommend creating a new folder in scratch for the assembly, like transcriptome_assembly. The output folder for Trinity should also contain the program’s name to avoid auto-deletion (Trinity will error out if you don’t do this!), so create another folder, such as trinity.
Now you can run a scheduler script to run Trinity, such as the one below.
#!/bin/bash -l
#PBS -m abe
#PBS -M your_email_adress@griffith/griffithuni.edu.au
#PBS -N trinity
#PBS -l select=4:ncpus=4:mpiprocs=4:mem=8gb,walltime=100:00:00
cd $PBS_O_WORKDIR
module load anaconda3/2021.11
source activate labmcdougallRoot
Trinity --seqType fq --trimmomatic --no_normalize_reads --jaccard_clip --max_memory 32G --CPU 4 --inchworm_cpu 4 --SS_lib_type RF --left ~/scratch/transcriptome_raw/RNA1_1.fastq.gz --right ~/scratch/transcriptome_raw/RNA1_2.fastq.gz --output ~/scratch/transcriptome_assembly/trinity
Under construction