transXpress: a Snakemake pipeline for streamlined de novo transcriptome assembly and annotation

Intro

Dependencies

transXpress requires:

• snakemake 5.4.2+ (install via conda, envs/default.yaml)
• fastqc (install via conda, envs/qc.yaml)
• multiqc (install via conda, envs/qc.yaml)
• trimmomatic (install via conda, envs/trimmomatic.yaml)
• Trinity (install via conda, trinity_utils.yaml)
• SPAdes (install via conda, envs/rnaspades.yaml)
• TransDecoder (install via conda, transdecoder.yaml)
• BioPython (install via conda, envs/default.yaml)
• samtools (install via conda, envs/trinity_utils.yaml)
• bowtie2 (install via conda, envs/trinity_utils.yaml)
• infernal (install via conda, envs/rfam.yaml)
• HMMER (install via conda, envs/pfam.yaml)
• kallisto (install via conda, envs/trinity_utils.yaml)
• NCBI BLAST+ (install via conda, envs/blast.yaml)
• R (install via conda, envs/trinity_utils.yaml)
• edgeR (install via conda, envs/trinity_utils.yaml)
• seqkit (install via conda, envs/default.yaml, envs/rnaspades.yaml)
• wget (install via conda, envs/default.yaml)
• sra-tools (install via conda, envs/default.yaml)
• tidyverse (required for Trinity, install via conda, envs/trinity_utils.yaml)
• python, numpy, pip (install via conda, envs/default.yaml)
• busco 4+ (install via conda, envs/busco.yaml)
• rsem (install via conda, envs/trinity_utils.yaml)
• SignalP 6.0
• TargetP 2.0
• tmhmm.py (install via pip, envs/default.yaml)
• basic Linux utitilies: split, awk, cut, gzip

The conda dependencies are installed in smaller conda environments automatically by transXpress (based on yaml files in the envs directory).

Installation

1. Checkout the transXpress code into the folder in which you will be performing your assembly:

git clone https://github.com/transXpress/transXpress.git

2. Install Mambaforge

curl -L -O "https://github.com/conda-forge/miniforge/releases/latest/download/Mambaforge-Linux-x86_64.sh"
bash Mambaforge-Linux-x86_64.sh
rm Mambaforge-Linux-x86_64.sh

3. To ensure correct versions of R packages will be used unset R_LIBS_SITE

unset R_LIBS_SITE

4. Setup main transXpress conda environment:

mamba activate base
mamba create -c conda-forge -c bioconda --name transxpress
mamba activate transxpress
conda config --set channel_priority disabled
mamba env update --file envs/default.yaml

• (TIP: if you have a problem updating the default environment try putting python 3.9 into the defaults.yaml file)

6. Create a tab-separated file called samples.txt in the assembly directory describing where to find your raw read FASTQ files. Create this file with the following contents:

   cond_A      cond_A_rep1 A_rep1_left.fq    A_rep1_right.fq
   cond_A      cond_A_rep2 A_rep2_left.fq    A_rep2_right.fq
   cond_B      cond_B_rep1 B_rep1_left.fq    B_rep1_right.fq
   cond_B      cond_B_rep2 B_rep2_left.fq    B_rep2_right.fq
   

• You can download reads from SRA with provided script:

  ./sra_download.sh <SRR0000000> <SRR0000001> <...>
  

  where SRR0000000 is an SRA readset ID. E.g., SRR3883773

• See the tests directory for an example of a samples file: test_samples.txt

7. Take a look at the configuration file config.yaml and update as required (you should check: assembler, targetp, signalp, strand_specific, lineage).

8. Setup other conda environments (This will take a while):

snakemake --use-conda --conda-frontend mamba --conda-create-envs-only --cores 10

9. Install SignalP 6.0 (fast):

   • Download SignalP 6.0 fast from https://services.healthtech.dtu.dk/services/SignalP-6.0/ (go to Downloads)
   • Unpack and install signalp:

      mamba env create -f envs/signalp.yaml
      mamba activate signalp
      tar zxvf signalp-6.0h.fast.tar.gz
      cd signalp6_fast
      pip install signalp-6-package/
      SIGNALP_DIR=$(python -c "import signalp; import os; print(os.path.dirname(signalp.__file__))" )
      cp -r signalp-6-package/models/* $SIGNALP_DIR/model_weights/
      mamba deactivate
     

     (make sure the conda python is used, or use the full path to python from your conda installation)

10. Install TargetP 2.0:

    • Download TargetP 2.0 from https://services.healthtech.dtu.dk/software.php
    • extract the tarball and add path to targetp /bin/ folder to the PATH variable

       mamba env create -f envs/targetp.yaml
       mamba activate targetp
       tar zxvf targetp-2.0.Linux.tar.gz
       export PATH=$PATH:`pwd`/targetp-2.0/bin
       mamba deactivate
      

Running transXpress

There are several options on how to run transXpress

Option 1 - use the provided script:

./transXpress.sh

Option 2 - run snakemake manually with 10 local threads:

snakemake --conda-frontend conda --use-conda --cores 10

Option 3 - run snakemake manually on an LSF cluster:

snakemake --conda-frontend conda --use-conda --latency-wait 60 --jobs 10000 --cluster 'bsub -n {threads} -R "rusage[mem={params.memory}000] span[hosts=1]" -oo {log}.bsub'

Option 4 - define a profile and run snakemake with the profile:

snakemake --profile profiles/ "$@"

You can find example of a simple profile config.yaml for slurm in the profiles directory or here

Running specific steps

You can run specific steps of the pipeline by specifying a rule or a resulting file:

# run only the rules until the multiqc_before_trim rule to check quality of the input data
./transXpress.sh multiqc_before_trim

# run only the rules to produce samples_trimmed.txt file
./transXpress.sh samples_trimmed.txt

or when using profiles:

# run only the rules until the multiqc_before_trim rule to check quality of the input data
snakemake multiqc_before_trim --profile profiles/ "$@"

# run only the rules to produce samples_trimmed.txt file
snakemake samples_trimmed.txt --profile profiles/ "$@"

Running tests

cd tests
./run_test.sh

Align reads to the transcriptome assembly and visualize the results in IGV

If you want to align reads to the transcriptome assembly and visualize the results in IGV, you can use the following commands:

./transXpress.sh align_reads
./transXpress.sh IGV

Then you can load your transcriptome file to IGV: Genomes -> Load Genome from File -> select the file transcriptome.fasta

Your sorted .bam files will be in the bowtie_alignments folder:

bowtie_alignments/{sample}.sorted.bam
bowtie_alignments/{sample}.sorted.bam.bai

Load them to IGV: File -> Load from File -> select the bowtie_alignments/{sample}.sorted.bam files

The directed acyclic graph (DAG) of the transXpress pipeline execution

Possible problems when executing on cluster systems

Time limit

Depending on the setup of your cluster you may have to add time option in the transXpress.sh script. If default time (depends on your cluster setup) for submitted job is not sufficient the job may be cancelled due to time limit.

If this is the case, add time option in submission command in transXpress.sh script.

For example, in case of Slurm change

snakemake --conda-frontend conda --use-conda --latency-wait 60 --restart-times 1 --jobs 10000 --cluster "sbatch -o {log}.slurm.out -e {log}.slurm.err -n {threads} --mem {params.memory}GB" "$@"

to

snakemake --conda-frontend conda --use-conda --latency-wait 60 --restart-times 1 --jobs 10000 --cluster "sbatch -o {log}.slurm.out -e {log}.slurm.err -n {threads} --mem {params.memory} --time=06:00:00" "$@"

This sets time limit to 6 hours. You may have to use different time limit based on size of reads used for assembly.

See https://github.com/trinityrnaseq/trinityrnaseq/wiki/Trinity-Computing-Requirements

Pipeline hangs when cluster cancels the job

It is possible that cluster cancels the job, but pipeline seems to be still running. This can happen because the pipeline does not receive information whether cluster job completed successfully, failed or is still running. You can add --cluster-status option and add script which detects the job status.

See https://snakemake.readthedocs.io/en/stable/tutorial/additional_features.html#using-cluster-status

Alternatively, you can use snakemake profiles which also contain status checking script.

See https://snakemake.readthedocs.io/en/v5.1.4/executable.html#profiles