How to create a workflow 

RattleSNP allows you to build a workflow using a simple config.yaml configuration file :

First, provide the data paths
Second, activate tools from mapping to SNP calling.
And last, manage parameters tools.

To create this file, just run:

create_config 

Create config.yaml for run

rattleSNP create_config [OPTIONS]

Options

-c, --configyaml <configyaml>: Required Path to create config.yaml

Then, edit the relevant sections of the file to customize your flavor of a workflow.

For FASTQ, naming convention accepted by RattleSNP is NAME_R1.fastq.gz or NAME_R1.fq.gz or NAME_R1.fastq or NAME_R1.fq. Preferentially use short names and avoid special characters because report can fail. Avoid to use the long name given directly by sequencer. Same for _R2 All fastq files have to be homogeneous on their extension and can be compressed or not.

Reference fasta file need a .fasta or .fa extension uncompressed.

2. Providing params 

PARAMS:
    MITOCHONDRIAL_NAME : ""
    # The filter suffix to add on vcf filter in order to allow multiple filter
    FILTER_SUFFIX: ["-Q30-DP5-MAF005-MISS07",
                    "-Q30-DP20-MAF001-MISS05"]

Find here a summary table with description of each params for RattleSNP :

Params	Description
MITOCHONDRIAL_NAME	The name of mitochondrial sequence on fasta, used to remove on VCF file. If not keep empty
FILTER_SUFFIX	The suffix name add to vcf filters file

3. Provide workflow step 

Activate/deactivate tools as you wish. Feel free to activate only assembly, assembly+polishing or assembly+polishing+correction.

Example:

################################
# Pipeline tools activation
FASTQC: true
CLEANING:
    ATROPOS: true
MAPPING:
    ACTIVATE: true
    TOOL: "BWA_MEM"         # Use BWA_MEM or BWA_SAMPE only
    BUILD_STATS: true      # warning if true but mapping false, mapping automatically run
SNPCALLING: true
FILTER: true                # Must be true if want run raxml or raxml-ng
RAXML: true

4. Parameters for some specific tools 

You can manage tools parameters on the params section in the config.yaml file.

Here you find standard parameters used on RattleSNP. Feel free to adapt it to your requires.

################################
# Misc. options for programs
PARAMS_TOOLS:
    ATROPOS: "--minimum-length 35  -q 20,20  -U 8  -O 10"
    FASTQC: ""
    BWA_ALN: ""
    BWA_SAMPE: ""
    BWA_MEM: ""
    SAMTOOLS_VIEW: "-bh -f 2"
    SAMTOOLS_SORT: ""
    SAMTOOLS_DEPTH: ""
    PICARDTOOLS_MARK_DUPLICATES: "-CREATE_INDEX TRUE -VALIDATION_STRINGENCY SILENT"
    GATK_HAPLOTYPECALLER: "--java-options '-Xmx40G' --emit-ref-confidence GVCF --output-mode EMIT_ALL_ACTIVE_SITES -ploidy 1"
    GATK_GENOMICSDBIMPORT: "--java-options '-Xmx40G' "
    GATK_GENOTYPEGVCFS: "--java-options '-Xmx40G' -new-qual"
    VCFTOOLS: ["--minDP 5 --minQ 30 --remove-indels --recode --recode-INFO-all --maf 0.05 --max-missing 0.7",
               "--minDP 20 --minQ 30 --remove-indels --recode --recode-INFO-all --maf 0.01 --max-missing 0.5"]
    RAXML: "-m GTRGAMMAX -f a -x $RANDOM -# autoMRE -p 600"
    RAXML_NG: "--all --model GTR+G --tree pars{50},rand{50} --bs-trees 100 --seed $RANDOM"

Warning

Please check documentation of each tool (outside of RattleSNP, and make sure that the settings are correct!)

How to run the workflow 

Before attempting to run rattleSNP, please verify that you have already modified the config.yaml file as explained in 1. Providing data.

If you installed RattleSNP on a HPC cluster with a job scheduler, you can run:

run_cluster 

Run snakemake command line with mandatory parameters.
SNAKEMAKE_OTHER: You can also pass additional Snakemake parameters using snakemake syntax.
These parameters will take precedence over Snakemake ones, which were defined in the profile.
See: https://snakemake.readthedocs.io/en/stable/executing/cli.html
Example:
rattleSNP run_cluster -c config.yaml –dry-run –jobs 200

rattleSNP run_cluster [OPTIONS] [SNAKEMAKE_OTHER]...

Options

-c, --config <config>: Required Configuration file for run tool

-pdf, --pdf

Run snakemake with –dag, –rulegraph and –filegraph

Default:: False

Arguments

SNAKEMAKE_OTHER: Optional argument(s)

run_local 

Run snakemake command line with mandatory parameters.
SNAKEMAKE_OTHER: You can also pass additional Snakemake parameters using snakemake syntax.
These parameters will take precedence over Snakemake ones, which were defined in the profile.
See: https://snakemake.readthedocs.io/en/stable/executing/cli.html
Example:
rattleSNP run_local -c config.yaml –threads 8 –dry-run
rattleSNP run_local -c config.yaml –threads 8 –singularity-args ‘–bind /mnt:/mnt’

rattleSNP run_local [OPTIONS] [SNAKEMAKE_OTHER]...

Options

-c, --config <config>: Required Configuration file for run tool

-t, --threads <threads>: Required Number of threads

-p, --pdf: Run snakemake with –dag, –rulegraph and –filegraph

Arguments

SNAKEMAKE_OTHER: Optional argument(s)

Advance run 

Providing more resources 

If the cluster default resources are not sufficient, you can edit the cluster_config.yaml file. See 2. Adapting cluster_config.yaml:

edit_cluster_config

Edit cluster_config.yaml use by profile

rattleSNP edit_cluster_config [OPTIONS]

Providing your own tools_config.yaml 

To change the tools used in a RattleSNP workflow, you can see 3. How to configure tools_path.yaml

edit_tools

Edit own tools version

rattleSNP edit_tools [OPTIONS]

Options

-r, --restore

Restore default tools_config.yaml (from install)

Default:: False

Output on RattleSNP 

The architecture of RattleSNP output is designed as follow:

OUTPUT_RattleSNP/
├── 1_mapping
├── 2_snp_calling
├── 3_full_snp_calling_stats
├── 4_raxml
├── LOGS

Report 

RattleSNP generates a useful report containing, foreach fastq, a summary of interesting statistics !!

Input	Description
FASTQ	Every paired FASTQ file should contain the whole set of reads to be mapped. Each fastq file will be mapped independently.
VCF	If SNP calling already run, you can use directly vcf to filter
REFERENCE_FILE	Only one REFERENCE genome file will be used by RattleSNP. This REFERENCE will be used for Mapping step
OUTPUT	output path directory