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Example data

Once everything is installed as outlined in the README document, you should be able to run the tutorial on some example data. The example can be run following method 2 (submitting a script) or method 3 (running the Snakemake pipeline). However, before running the example, you will need to download the example data from Zenodo. We include a chromosome from our naked mole-rat assembly (plus RNA-seq and ISO-seq data) to try this tutorial. We assume that the following wget command will be performed in /path-to-GAT/GenAnT (see /setup/GAT-InstallAndDownload.md for details).

wget https://zenodo.org/records/14962941/files/example_data.tar.gz
tar -xvzf example_data.tar.gz

You will also need the reference genome for the naked mole-rat. In /path-to-GenomeAnnotationTutorial/GenAnT/data/references (make this directory if it doesn't exist):

mkdir -p HetGlaV2_female ; cd HetGlaV2_female

wget https://ftp.ensembl.org/pub/release-115/fasta/heterocephalus_glaber_female/dna/Heterocephalus_glaber_female.Naked_mole-rat_maternal.dna_sm.toplevel.fa.gz
wget https://ftp.ensembl.org/pub/release-115/gff3/heterocephalus_glaber_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.gff3.gz
for i in *.gz ; do gunzip $i ; echo $i ; done
  bash ../../../setup/reference_directory_ensembl.sh \
  . \ # path to the reference genome directory
  ~/GenAnT \ #  path to GenomeAnnotationTutorial ( `GenAnT` included)
  Heterocephalus_glaber_female.Naked_mole-rat_maternal.dna_sm.toplevel.fa \
  Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.gff3
cd ..

And then we will need a mouse reference genome, of course, for running TOGA. For this example, we'll use the GRCm39 RefSeq genome. This can also be done in the references directory:

mkdir -p mmus_GRC39 ; cd mmus_GRC39

for i in *.gz ; do gunzip $i ; echo $i ; done
  bash ../../../setup/reference_directory_refseq.sh \
  . \
  ~/GenAnT \
  GCF_000001635.27_GRCm39_genomic.fna \
  GCF_000001635.27_GRCm39_genomic.gff

Method 2 (script submission)

Assuming everything is properly set up, running the tutorial without flow control involves submitting the execute script with two positional arguments: bash Execute_GAT_in_serial.sh path-to-GAT path-to-Conda For us this looks like

bash Execute_GAT_in_serial.sh \
/.mounts/labs/simpsonlab/users/dsokolowski/projects/GenAnT \
/.mounts/labs/simpsonlab/users/dsokolowski/miniconda3

Even annotating one chromosome is relatively resource intensive, so we reccomend submitting this as a job (Recommended: 64G mem, 16 cores, 72h runtime)

Lastly, this script has a module load singularity. If you access singularity differently (e.g., apptainer, conda environment etc.) then replace that line with what you need to have singularity accessible.

Method 3 (Snakemake)

Running the example data using the snakemake pipeline takes slightly more work:

There is a config file (config_example.yaml) located in the GenAnT_Snakemake directory which will need to be modified to run the example data. To run the tutorial with the example data, you need to change: "/path-to-conda/miniconda3/" to the path to the miniconda directory where the annotation_tutorial environment lives (e.g., /.mounts/labs/simpsonlab/users/dsokolowski/miniconda3/) and change "path-to-GenAnT/" to the path where you cloned "GenAnT" (e.g., /.mounts/labs/simpsonlab/users/dsokolowski/projects/GenAnT). This will have to be done for each line in config_example.yaml that uses the GenAnT file path. Let's walk through this line by line.

Here is what the example configuration file looked like on my computer after I changed all the variables:

sourceDir: "/opt/miniconda3/bin/activate"
externalDir: "/home/baderlab/zclarke/GenAnT/external
dataDir: "/home/baderlab/zclarke/GenAnT/data"

# Parameters describing your assembly + annotation
outDir: "/scratch8/badertmp/GenAnT_Example_stranded"
target: "example"
species: "heterocephalus_glaber"

assemblyFile: "/home/baderlab/zclarke/GenAnT/data/example_data/NMRchr28.fa"
MaskedAssemblyFile: "none"
MaskedAssemblyAnnotation: "none"
rnaseqDir: "/home/baderlab/zclarke/GenAnT/data/example_data/RNAseq_alignment"
isoseqDir: "/home/baderlab/zclarke/GenAnT/data/example_data/ISOseq_alignment"
customRef: "FALSE"
liftoffRef: "FALSE"

# Parameters describing your reference assemblies + annotation

refToga: "mouse"
TogaDir: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39"
refTogaFa: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39/GCF_000001635.27_GRCm39_genomic.fna"
refTogaBed: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39/GCF_000001635.27_GRCm39_genomic.toga.bed"
refTogaIsoform: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39/GCF_000001635.27_GRCm39_genomic.isoforms.tsv"

refToga2: "NMR"
TogaDir2: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female"
refTogaFa2: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.clean.fa"
refTogaBed2: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.clean.bed"
refTogaIsoform2: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.clean.isoforms.tsv"

refLiftOff: "NMR"
refLiftOffFa: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.clean.fa"
refLiftOffGff: "/home/baderlab/zclarke/GenAnT/data/references/HetGlaV2_female/Heterocephalus_glaber_female.Naked_mole-rat_maternal.115.gffread.gff"

orthofinderFA: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39/GCF_000001635.27_GRCm39_genomic.nostop.protein.faa"
orthofinderTab: "/home/baderlab/zclarke/GenAnT/data/references/mmus_GRC39/GCF_000001635.27_GRCm39_genomic.table.txt"

# Tool specific parameters
dfamDB: "rodentia"
brakerOdbFaa: "Vertebrata.fa"
mikadoScore: "mammalian.yaml"
mirmachineClade: "Mammalia"
stranded: "TRUE"
rnaseqCov: 5
isoseqCov: 1
stringtieMerge: "FALSE"

# Adding GFF files that you have already computed
customGFF: "none"
customLiftoff: "none"
customTOGA: "none"
customTOGA2: "none"
customBraker: "none"
customStringtie: "none"

Once the configuration file is made, Snakemake can be run from the GenAnT_Snakemake directory according to your scheduler. Here are a couple of examples of the line that can be used to run snakemake:

snakemake --configfile config_example.yaml --jobs 750 --latency-wait 60 --cluster "qsub -cwd -V -o snakemake.output.log \
 -e snakemake.error.log -pe smp {threads} -l h_vmem={params.memory_per_thread} \
 {params.extra_cluster_opt} -l h_stack=32M -l h_rt={params.walltime} -P simpsonlab -b y" "$@"

or the following for a SLURM scheduler

snakemake --configfile config_example.yaml  --jobs 750 --latency-wait 60 \
 --cluster "sbatch --cpus-per-task={threads} --mem-per-cpu={params.memory_per_thread} \
 --time={params.walltime} --output=logs/{rule}.%j.out --error=logs/{rule}.%j.err"

Snakemake uses the "Snakefile" that's in the GenAnT_Snakemake directory for both the example run and a real run, and this dictates the memory and threading used by Snakemake. If Snakemake fails due to such an issue, the memory and/or threads defined by the rules in the Snakefile may have to be reduced to match your cluster's resources. This will extend the time required to run the example.