Profile Options & Input Files

This section walks through the available parameters to customize your workflow.

4.1 Input Files Required:

(A) This table lists the required files to run metadata validation and annotation:

Input files	File type	Description
fasta	.fasta	Single sample fasta sequence file(s)
metadata	.xlsx	Multi-sample metadata matching metadata spreadsheets provided in input_files
ref_fasta	.fasta	Reference genome to use for the liftoff_submission branch of the pipeline
ref_gff	.gff	Reference GFF3 file to use for the liftoff_submission branch of the pipeline

** Please note that the pipeline expects ONLY pre-split FASTA files, where each FASTA file contains only the sequence(s) associated with its corresponding sample. The name of each FASTA file corresponding to a particular sample must be placed within your metadata sheet under fasta_file_name.

Here is an example of how this would look like.

(B) This table lists the required files to run with submission:

Input files	File type	Description
fasta	.fasta	Single sample fasta sequence file(s) sequences
metadata	.xlsx	Multi-sample metadata matching metadata spreadsheets provided in input_files
ref_fasta	.fasta	Reference genome to use for the liftoff_submission branch of the pipeline
ref_gff	.gff	Reference GFF3 file to use for the liftoff_submission branch of the pipeline
submission_config	.yaml	configuration file for submitting to NCBI, sample versions can be found in repo

4.2 Customizing Parameters:

Parameters can be customized during runtime by providing various flags after the nextflow command.

Example: Modifying the path of the output directory

nextflow run main.nf -profile test,singularity --virus --output_dir /path/to/output/dir

The complete list of parameters and the types of input that they require can be found under in the Parameters page.

4.3 Understanding Profiles and Environments:

Within the nextflow pipeline the -profile option is required as an input. The profile option can be specified as test. If test is not specified, the standard profile will be run as the default. The test params should remain the same for testing purposes, but the standard profile can be changed to fit user preferences. The run environment is supplied as the second arugument to the profile parameter. The options of docker, singularity or conda can passed in. As conda is unstable, we recommend you choose singularity or docker.

4.4 Toggling Submission:

Now that your file paths are set within your standard.yml or standard.json or standard_params.config file, you will want to define whether to run the full pipeline with submission or without submission. This is defined within the standard_params.config file underneath the subworkflow section as run_submission run_submission = true/false

4.5 More Information on Submission:

The submission component of the pipeline uses the processes that are directly integrated from SeqSender public database submission pipeline. It has been developed to allow the user to create a config file to select which databases they would like to upload to and allows for any possible metadata fields by using a YAML to pair the database's metadata fields with your personal metadata field columns. The requirements for this portion of the pipeline to run are listed below.

(A) Create Appropriate Accounts as needed for the SeqSender public database submission pipeline integrated into TOSTADAS:

• NCBI: If uploading to NCBI archives such as BioSample/SRA/Genbank, you must complete the following steps:

  1. Create a center account: Contact the following e-mail for account creation : [email protected] and provide the following infomation:
     • Suggested center abbreviation (16 char max)
     • Center name (full), center URL & mailing address (including country and postcode)
     • Phone number (main phone for center or lab)
     • Contact person (someone likely to remain at the location for an extended time)
     • Contact email (ideally a service account monitored by several people)
     • Whether you intend to submit via FTP or command line Aspera (ascp)
  2. Gain access to an upload directory: Following center account creation, a test area and a production area will be created. Deposit the XML file and related data files into a directory and follow the instructions SRA provides via email to indicate when files are ready to trigger the pipeline.

• GISAID: A GISAID account is required for submission to GISAID, you can register for an account at https://www.gisaid.org/. Test submissions are first required before a final submission can be made. When your first test submission is complete contact GISAID at [email protected] to recieve a personal CID. GISAID support is not yet implemented but it may be added in the future.

(B) Config File Set-up:

• The template for the submission config file can be found in bin/default_config_files within the repo. This is where you can edit the various parameters you want to include in your submission. Read more at the SeqSender docs.
• You can find more information on how to setup your own submission config and additional information on fields in the following guide: Submission Config Guide.

❗ Pre-requisite to submit to GenBank: Copy the program table2asn to you tostadas/bin directory by running the following lines of code:

cd ./tostadas/bin/
wget https://ftp.ncbi.nlm.nih.gov/asn1-converters/by_program/table2asn/linux64.table2asn.gz
gunzip linux64.table2asn.gz
mv linux64.table2asn table2asn