RNA-Seq to BUSCO - Workflow

Workflow to quality-check RNA-Seq data prior to and post trimming with Trimmomatic. Reads are assembled with two de novo transcriptome assemblers, Trinity and Shannon, but more will be added soon. BUSCO is used to annotate and benchmark the assemblies.

To be implemented: Inclusion of tool to select the best annotated assembly based on the results from BUSCO, Ortho-Overlap.

System requirements

Local machine

I recommend running the workflow on a HPC system, as the analyses are resource and time consuming.

• If you don't have it yet, it is necessary to have conda or miniconda in your machine. Follow there instructions.

  • After you are all set with conda, I highly (highly!) recommend installing a much much faster package manager to replace conda, mamba

    • First activate your conda base:

    conda activate base

    • Then, type:

    conda install -n base -c conda-forge mamba

• Likewise, follow this tutorial to install Git if you don't have it.

HPC system

Follow the instructions from your cluster administrator regarding loading of modules, such as loading a root distribution from Conda. For example, with the cluster I work with, we use modules to set up environmental variables, which have to first be loaded within the jobscripts. They modify the $PATH variable after loading the module.

e.g.: module load anaconda3/2022.05

You usually don't have sudo rights to install anything to the root of the cluster. So, as I wanted to work with a more updated distribution of conda and especially use mamba to replace conda as a package manager, I had to first create my own "local" conda, i.e. I first loaded the module and then created a new environment I called localconda

1. module load anaconda3/2022.05
2. conda create -n localconda -c conda-forge conda=22.9.0
3. conda install -n localconda -c conda-forge mamba
4. conda activate localconda

If you run conda env list you'll probably see something like this: /home/myusername/.conda/envs/localconda/

Data requirements

1. Paired-end reads (Forward and Reverse)

Move your RNA-Seq data into resources/raw_data

Note: Right now, the workflow only works with species which have paires-end reads. This will modified later. Likewise, a system to automate the SRA data download from NCBI will be implemented

2. Tab-separates, species table

Template table provided in config/species_table.tsv. Modify following the name of your species and the filenames from the paired-end reads.

This is important not only to know how the raw data is named, but also to write the names of the files in meaninful ways, i.e. HW03_Berthella_plumula.fasta instead of something like SRR8573930.fasta. Also really important for the final graph from busco report.

Important:

• No cell can be empty, as Snakemake will see this as missing input file and the analyses will not run
• Never modify the headers from the table otherwise the same thing will happen
• The names of the forward and reverse files have to be the same as the actual files you copied into resources/raw_data

Species_name	Forward	Reverse
HW03_Berthella_plumula	SRR8573930_1.fastq.gz	SRR8573930_2.fastq.gz
My_species	Myspecies_S1_L001_R1_001.fastq.gz	Myspecies_S1_L001_R2_001.fastq.gz

3. Config file

Template found in config/configfile.yaml. Modify accordingly. Required file for important settings from the analyses. Workflow will fail if anything is wrong or missing.

• sample_info: you can keep this path, but remember to modify the information according to your samples (See previous step in the data requirements)
• max_memory: max amount of memory Trinity assembler can use
• adapter: adpater file used by Trimmomatic. You can provide a custom adapter file, like is the case in the template file, or the name from one of the Fasta files supplied by Trimmomatic. The most common one is TruSeq3-PE.fa, as it is used for by HiSeq and MiSeq machines. See page 12 from the Trimmomatic manual for more information.
• lineage: clade-specific information to identify BUSCO genes in the transcripts. Click here for all BUSCO lineages.

Installation

1. Clone this repository

git clone https://gitlab.leibniz-lib.de/jwiggeshoff/rna-seq-to-busco.git

2. Activate your conda base

conda activate base

• If you are working on a cluster or have your own "local", isolated environment you want to activate instead (see here), use its name to activate it

conda activate localconda

3. Install rna-seq-to-busco into an isolated software environment by navigating to the directory where this repo is and run:

conda env create --file environment.yaml

If you followed what I recommended in the System requirements, run this intead:

mamba env create --file environment.yaml

The environment from rna-seq-to-busco is created

4. Always activate the environment before running the workflow

On a local machine:

conda activate rna-seq-to-busco

If you are on a cluster and/or created the environment "within" another environment, you want to run this first:

conda env list

You will probably see something like this among your enviornments:

home/myusername/.conda/envs/localconda/envs/rna-seq-to-busco

From no own, you have to give this full path when activating the environment prior to running the workflow

conda activate /home/myusername/.conda/envs/localconda/envs/rna-seq-to-busco

Starting the workflow

Remember to always activate the environment first

conda activate rna-seq-to-busco

or

conda activate /home/myusername/.conda/envs/localconda/envs/rna-seq-to-busco

Local machine

Not recommended if you don't have a lot of storage and CPUs available (and time to wait...). Nevertheless, you can simply run like this:

nohup snakemake --keep-going --use-conda --verbose --printshellcmds --reason --nolock --cores 11 > nohup_$(date +"%F_%H").out &

Modify number of cores accordingly.

HPC system

This option was tested to run the workflow in HPC clusters using the Sun Grid Engine (SGE) queue scheduler system. For other systems, read more here.

Before the first execution of the workflow

Run this to create the environments from the rules:

snakemake --cores 8 --use-conda --conda-create-envs-only

Then:

nohup snakemake --configfile config/configfile.yaml --keep-going --use-conda --verbose --printshellcmds --reason --nolock --rerun-incomplete --jobs 15 --cores 41 --local-cores 15 --max-threads 25 --cluster "qsub -terse -V -b y -j y -o snakejob_logs/ -cwd -q fast.q,small.q,medium.q,large.q -M [email protected] -m be -pe smp {threads}" > nohup_rna-seq-to-busco_$(date +"%F_%H_%M_%S").out &

Remember to:

1. Create snakejob_logs in the working directory: mkdir -p snakejob_logs
2. Modify [email protected]
3. Change values for --jobs, --cores, --local-cores, and --max-threads accordingly
   • Important: Make sure you set a low value for --local-cores to not take up too much resources from your host node

Finishing the workflow: report.zip

Upon successfully finishing the analyses, Snakemake will automatically generate a compressed report in the working directory, report.zip.

It describes the used software versions, the reports from FastQC, as well as the final graph generated by the built-in BUSCO report. It also includes the commands and paths to in and output files.

To know more, see the documentation from Snakemake here.

Done :)