RefNAAP-wdl

WDL wrapper around RefNAAP and RABV-GLUE for execution on Terra.bio. Additionally, a custom Coding DNA Sequence (CDS) coverage calculation module is available for quality-control of the processed data.

Figure 1: RefNAAP-wdl.

Workflow components

What is RefNAAP?

RefNAAP is a reference based Oxford Nanopore Technologies (ONT) assembly analysis pipeline for RABV genomes. In summary, it performs the following steps:

1. It QCs the files using fastQC and multiQC to generate a quality report.
2. It trims the left and right ends of the reads by 25 basepairs, and filters out reads shorter than 50bp. These values can be customized.
3. It generates the assembly reads using reference-based assembly with minimap2, gap fixing, and medaka.

It uses a reference file composed of 14 different RABV sequences for the reference-based assembly.

What is RABV-GLUE?

RABV-GLUE is a sequence-oriented resource for comparative genomic analysis of rabies virus (RABV), developed using the GLUE software framework. It organises RABV genome sequence data along evolutionary lines, aiming to leverage new and existing RABV sequences in order to improve our understanding of the epidemiology and pathology of RABV. It provides the following information:

1. RABV Major Clade
2. RABV Minor Clade
3. Closest full genome accession in the RABV-GLUE database

What is the custom coverage module?

To emulate the behaviour of RABV-GLUE Online, a custom module was created that recieves the closest full genome accession and downloads it from NCBI. This is then used to create a [BLAST] database for the calculation of coverage per CDS. A sequence is idenfitied as RABV if at least one CDS has over 75 percent coverage (default value, it can be adjusted). It produces the following information:

1. Identification of RABV sequence
2. Per CDS coverage (N, P, M, G and L)

How do i use RefNAAP-wl on Terra?

RefNAAP-wdl is available on Terra.bio, a cloud-native platform for researchers to access data, run analysis tools, and collaborate. With Terra.bio, you can easily process your data without prior knowledge of the command-line.

The following steps, assume you have already set up an account on Terra.bio and created a workspace to work with RefNAAP-wdl.

Import the workflow from Dockstore

To begin using RefNAAP-wdl on Terra.bio, you will need to import the workflow from Dockstore, which is available at: RefNAAP-wdl Dockstore Import.

Figure 2: RefNAAP-wdl on Dockstore.

Once you are on the Dockstore page for RefNAAP-wdl, you will want to locate the Launch with section on the right side of the page and click on Terra.

Figure 3: Launching a workflow with Terra.bio on Dockstore.

After clicking the Terra button, you will be transported to Terra.bio. Once here you will decide on the Destination Workspace. Please select which of your workspaces you would like to import this workflow into. Once you have selected a Destination Workspace, all that remains is to click the Import button.

Figure 4: Importing workflow interface on Terra.bio.

Workflow configuration on Terra.bio

The RefNAAP-wdl should now be available in Terra.bio on the WORKFLOWS tab. When clicking on the RefNAAP-wdl the workflow interface loads. On the workflow configuration section you will need to select the Run workflow(s) with inputs defined by data table. RefNAAP-wdl is a sample-level workflow.

Figure 5: RefNAAP-wdl on Terra.bio.

Workflow inputs

Several inputs are available for workflow costumization: required inputs that are necessary for execution, and optional inputs that have default values but can be overwritten by the user.

Note: To provide inputs from the data table, terra uses the this.{column_name} notation. For example, to pass the ONT reads that are in the ont_read column on the data table to the read1 input, the value should be passed as this.ont_reads.

Table 1: Input description for RefNAAP-wdl

Terra Task Name	Variable	Type	Description	Default Value	Terra Status
refnaap_wf	read1	File	Base-called ONT read file in FASTQ file format (compressed).		Required
refnaap_wf	samplename	String	Name of sample to be analyzed.		Required
ncbi_datasets_blast	blast_evalue	String	BLAST e-value threshold.	"1e-10"	Optional
ncbi_datasets_blast	cpu	Int	Number of CPUs to allocate to the task.	4	Optional
ncbi_datasets_blast	disk_size	Int	Amount of storage (in GB) to allocate to the task.	50	Optional
ncbi_datasets_blast	docker	String	The Docker container to use for the task.	"us-docker.pkg.dev/general-theiagen/theiagen/ncbi-datasets-blast:16.38.1_20250321"	Optional
ncbi_datasets_blast	memory	Int	Amount of memory/RAM (in GB) to allocate to the task.	8	Optional
ncbi_datasets_blast	min_gene_coverage	Float	Minimum percent coverage for BLAST to determine the persence of a CDS.	75.0	Optional
ncbi_datasets_blast	min_percent_identity	Float	Minimum percent identity for BLAST to determine the persence of a CDS.	75.0	Optional
rabv_genotype	cpu	Int	Number of CPUs to allocate to the task.	4	Optional
rabv_genotype	disk_size	Int	Amount of storage (in GB) to allocate to the task.	50	Optional
rabv_genotype	docker	String	The Docker container to use for the task.	"us-docker.pkg.dev/general-theiagen/theiagen/rabvglue:1.1.113_20250320"	Optional
rabv_genotype	memory	Int	Amount of memory/RAM (in GB) to allocate to the task.	8	Optional
refnaap	cpu	Int	Number of CPUs to allocate to the task.	8	Optional
refnaap	disk_size	Int	Amount of storage (in GB) to allocate to the task.	100	Optional
refnaap	docker	String	The Docker container to use for the task.	"us-docker.pkg.dev/general-theiagen/internal/refnaap:b3ad097"	Optional
refnaap	memory	Int	Amount of memory/RAM (in GB) to allocate to the task.	16	Optional
refnaap	min_coverage	Int	Amplicon regions need a minimum of this average coverage number.	5	Optional
refnaap	model	String	Basecall model.	"r10_min_high_g303"	Optional
refnaap	size	Int	Filter reads less than this length.	50	Optional
refnaap	trim_left	Int	Bases to trim from left side of read.	25	Optional
refnaap	trim_right	Int	Bases to trim from right side of read.	25	Optional

Note: Available basecall models:

r103_min_high_g345, r103_min_high_g360, r103_prom_high_g360, r103_prom_snp_g3210, r103_prom_variant_g3210, r10_min_high_g303, r10_min_high_g340, r941_min_fast_g303, r941_min_high_g303, r941_min_high_g330, r941_min_high_g340_rle, r941_min_high_g344, r941_min_high_g351, r941_min_high_g360, r941_prom_fast_g303, r941_prom_high_g303, r941_prom_high_g330, r941_prom_high_g344, r941_prom_high_g360, r941_prom_high_g4011, r941_prom_snp_g303, r941_prom_snp_g322, r941_prom_snp_g360, r941_prom_variant_g303, r941_prom_variant_g322, r941_prom_variant_g360

Note: When BLASTing to calculate the percent coverage of a given CDS, only the largest fragment that alignes is considered.

Workflow outputs

The RefNAAP-wdl produces several outputs that are populated back to the data table.

Table 2: Output description for RefNAAP-wdl

Variable	Type	Description
blast_results	File	File containing the BLAST results.
datasets_ncbi_docker	String	Dockerfile used for the NCBI datasets module.
datasets_ncbi_reference_fasta	File	File, in FASTA format, with the closest full reference sequence identified by RABV-GLUE and used to create the BLAST database.
datasets_ncbi_report	File	Report file from NCBI Datasets for the reference sequence download.
datasets_ncbi_version	File	Version of NCBI Datasets used.
G_percent_coverage	Float	Percent coverage of the G CDS in the RABV genome.
L_percent_coverage	Float	Percent coverage of the L CDS in the RABV genome.
M_percent_coverage	Float	Percent coverage of the M CDS in the RABV genome.
N_percent_coverage	Float	Percent coverage of the N CDS in the RABV genome.
P_percent_coverage	Float	Percent coverage of the P CDS in the RABV genome.
rabv_identified	String	Indication that the sequence analysed has been identified as RABV.
rabvglue_closest_reference	String	Accession of the closest reference identified by RABV-GLUE.
rabvglue_major_clade	String	Major clade identified by RABV-GLUE.
rabvglue_minor_clade	String	Minor clade identified by RABV-GLUE.
refnaap_analysis_date	String	Date of analysis with RefNAAP.
refnaap_assembly_fasta	File	Consensus assembly generated by RefNAAP in FASTA format.
refnaap_docker	String	Dockerfile used for the RefNAAP module.
refnaap_multiqc_report	File	MultiQC report generated by RefNAAP in HTML format.

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RABVGlue-wdl

The RABV-GLUE and custom Coding DNA Sequence (CDS) coverage calculation modules of the RefNAAP-WDL workflow are also available as it's own standalone workdlow for execution on Terra.bio. This allows for the analysis of RABV genomes that have been assembled through alternative methods, such as de novo assembly.

How do i use RABVGlue-wl on Terra?

The following steps, assume you have already set up an account on Terra.bio and created a workspace to work with RABVGlue-wdl.

Import the workflow from Dockstore

To begin using RABVGlue-wdl on Terra.bio, you will need to import the workflow from Dockstore, which is available at: RABVGlue-wdl Dockstore Import.

Figure 6: RABVGlue-wdl on Dockstore.

Once you are on the Dockstore page for RABVGlue-wdl, you will want to locate the Launch with section on the right side of the page and click on Terra.

Figure 7: Launching a workflow with Terra.bio on Dockstore.

After clicking the Terra button, you will be transported to Terra.bio. Once here you will decide on the Destination Workspace. Please select which of your workspaces you would like to import this workflow into. Once you have selected a Destination Workspace, all that remains is to click the Import button.

Figure 8: Importing workflow interface on Terra.bio.

Workflow configuration on Terra.bio

The RABVGlue-wdl should now be available in Terra.bio on the WORKFLOWS tab. When clicking on the RABVGlue-wdl the workflow interface loads. On the workflow configuration section you will need to select the Run workflow(s) with inputs defined by data table. RABVGlue-wdl is a sample-level workflow.

Figure 5: RABVGlue-wdl on Terra.bio.

Workflow inputs

Several inputs are available for workflow costumization: required inputs that are necessary for execution, and optional inputs that have default values but can be overwritten by the user.

Note: To provide inputs from the data table, terra uses the this.{column_name} notation. For example, to pass the ONT reads that are in the ont_read column on the data table to the read1 input, the value should be passed as this.ont_reads.

Table 1: Input description for RefNAAP-wdl

Terra Task Name	Variable	Type	Description	Default Value	Terra Status
rabvglue_wf	assembly_fasta	File	FASTA file with the RABV sequence to be analyzed.		Required
ncbi_datasets_blast	blast_evalue	String	BLAST e-value threshold.	"1e-10"	Optional
ncbi_datasets_blast	cpu	Int	Number of CPUs to allocate to the task.	4	Optional
ncbi_datasets_blast	disk_size	Int	Amount of storage (in GB) to allocate to the task.	50	Optional
ncbi_datasets_blast	docker	String	The Docker container to use for the task.	"us-docker.pkg.dev/general-theiagen/theiagen/ncbi-datasets-blast:16.38.1_20250321"	Optional
ncbi_datasets_blast	memory	Int	Amount of memory/RAM (in GB) to allocate to the task.	8	Optional
ncbi_datasets_blast	min_gene_coverage	Float	Minimum percent coverage for BLAST to determine the persence of a CDS.	75.0	Optional
ncbi_datasets_blast	min_percent_identity	Float	Minimum percent identity for BLAST to determine the persence of a CDS.	75.0	Optional
rabv_genotype	cpu	Int	Number of CPUs to allocate to the task.	4	Optional
rabv_genotype	disk_size	Int	Amount of storage (in GB) to allocate to the task.	50	Optional
rabv_genotype	docker	String	The Docker container to use for the task.	"us-docker.pkg.dev/general-theiagen/theiagen/rabvglue:1.1.113_20250320"	Optional
rabv_genotype	memory	Int	Amount of memory/RAM (in GB) to allocate to the task.	8	Optional

Note: When BLASTing to calculate the percent coverage of a given CDS, only the largest fragment that alignes is considered.

Workflow outputs

The RABVGlue-wdl produces several outputs that are populated back to the data table.

Table 2: Output description for RefNAAP-wdl

Variable	Type	Description
blast_results	File	File containing the BLAST results.
datasets_ncbi_docker	String	Dockerfile used for the NCBI datasets module.
datasets_ncbi_reference_fasta	File	File, in FASTA format, with the closest full reference sequence identified by RABV-GLUE and used to create the BLAST database.
datasets_ncbi_report	File	Report file from NCBI Datasets for the reference sequence download.
datasets_ncbi_version	File	Version of NCBI Datasets used.
G_percent_coverage	Float	Percent coverage of the G CDS in the RABV genome.
L_percent_coverage	Float	Percent coverage of the L CDS in the RABV genome.
M_percent_coverage	Float	Percent coverage of the M CDS in the RABV genome.
N_percent_coverage	Float	Percent coverage of the N CDS in the RABV genome.
P_percent_coverage	Float	Percent coverage of the P CDS in the RABV genome.
rabv_identified	String	Indication that the sequence analysed has been identified as RABV.
rabvglue_closest_reference	String	Accession of the closest reference identified by RABV-GLUE.
rabvglue_major_clade	String	Major clade identified by RABV-GLUE.
rabvglue_minor_clade	String	Minor clade identified by RABV-GLUE.

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Contact

If you have any questions or concerns, please raise a GitHub issue or email Theiagen's general support at [email protected].