This repository contains two pipelines for processing SARS-CoV-2 data:
- The "from assembly" pipeline converts assembled SARS-CoV-2 genomes to variant calls.
- The "from fastq" pipeline converts from raw reads (fastqs) to variants and assembly files. This pipeline is able to process both Nanopore (single-end) and Illumina paired-end sequencing data.
Workflow to process assembly files to variants.
Required reference files can be downloaded using the download-data.sh script in the scripts directory by running:
./scripts/download-data.sh -aThis will download required data files to the data directory at the root of the repository.
Inputs must be selected and filled in to a JSON-format file. A template file may be found here: inputs/from_assembly/input_template.json.
| Input identifier | Description | Possible values |
|---|---|---|
| assembly_to_vcf.sample_id | Sample ID; Used to name output files | |
| assembly_to_vcf.assembly | SARS-CoV-2 assembled genome (.fa or .fasta) file | |
| assembly_to_vcf.reference_genome | Path to the SARS-CoV-2 viral reference FASTA (MN908947.3) | Downloaded using the scripts/download-data.sh script with the -a option. |
| assembly_to_vcf.reference_genome_id | MN908947.3 | |
| assembly_to_vcf.date | Date (added to the VCF) | Format YYYY-mm-dd_HH:MM:SS |
| assembly_to_vcf.snpEff_genome | snpEff genome identifier (NC_045512.2) | |
| assembly_to_vcf.snpEff_db | Path to the snpEff database used for annotation | Downloaded using the scripts/download-data.sh script with the -a option. |
- Example input file using an assembly from GenBank: inputs/from_assembly/test_input.json
| Output identifier | Description | Notes |
|---|---|---|
| annotated_vcf, annotated_vcf_index | Variant calls and index | While this file is in variant call format, these calls do not incorporate base quality scores and only represent a difference between the sample assembly and the reference genome. These calls should be carefully inpected and caution should be used in combining these variant calls with those called from raw data, where base quality information is available. |
Workflows to process fastq files to variants and assemblies.
This workflow wraps two separate analysis pipelines:
- The SARS-CoV-2 Illumina GeNome Assembly Line (SIGNAL) pipeline for Illumina paired-end data; created by the McArthur lab
- An implementation of the ARTIC protocol for use with Nanopore data by the Connor lab
Visit the repositories for each workflow for more information on the steps, protocols, and parameters in use.
Some reference files are required by the SIGNAL pipeline. They can be downloaded automatically using the download-data.sh script in the scripts directory by running:
./scripts/download-data.sh -fThis will download required data files to the data directory at the root of the repository. These files total ~4.5 GB.
Inputs must be selected and filled in to a JSON-format file. A template file may be found here: inputs/from_fastq/input_template.json.
| Input identifier | Description | Possible values |
|---|---|---|
| main.accession | NCBI run accession ID | Find SARS-CoV-2 sequencing runs here |
| main.type | Sequencing platform used; determines which analysis pipeline is run | Must be one of ["NANOPORE", "ILLUMINA_PE"] |
| main.signal_scheme_bed | [SIGNAL] The BED-format primer scheme used to prepare the Illumina PE library | Only used in the ILLUMINA_PE workflow (SIGNAL). Can be the path to any properly formatted file. See primer file directory and the section on primer schemes below. |
| main.signal_primer_pairs_tsv | [SIGNAL] Primer pair tsv file used for iVar's amplicon filter | Only used in the ILLUMINA_PE workflow (SIGNAL). See primer file directory and the section on primer schemes below. |
| main.signal_amplicon_bed | [SIGNAL] The BED-format amplicon locations (not the primers) | Only used in the ILLUMINA_PE workflow (SIGNAL). See primer file directory and the section on primer schemes below. |
| main.artic_primer_version | [ARTIC] The ARTIC primer version used to prepare the Nanopore library | Only used in the NANOPORE workflow (ARTIC). Must be one of ["V1", "V2", "V3"]. Other primer schemes for Nanopore samples are not currently supported. |
| main.composite_reference | [SIGNAL] Path to an archive file containing human reference sequences | Downloaded using the scripts/download-data.sh script with the -f option. |
| main.kraken_db | [SIGNAL] Path to an archive file containing the Kraken2 database files | " " " |
| main.viral_reference_genome | [SIGNAL] Path to the SARS-CoV-2 viral reference FASTA (MN908947.3) | " " " |
| main.viral_reference_genome | [SIGNAL] Path to the SARS-CoV-2 viral reference FASTA (MN908947.3) | " " " |
| main.viral_reference_contig_name | Name to set the SARS-CoV-2 contig name to (MN908947.3) | |
| main.min_freq_high_confidence_threshold | [SIGNAL] Frequency threshold to call high-confidence variants | Default: 0.25 |
| main.ivar_min_freq_threshold | [SIGNAL] Minimum frequency threshold to call variants | Default: 0.03 |
| main.ivar_min_coverage_depth | [SIGNAL] Minimum coverage depth to call variants | Default: 10 |
| main.ivar_min_variant_quality | [SIGNAL] Minimum variant quality | Default: 20 |
| main.ivar_freq_threshold | [SIGNAL] Frequency threshold to build consensus sequence | Default: 0.75 |
| main.mpileup_depth | [SIGNAL] Mpileup depth for iVar | Default: 100000 |
| main.signal_min_qual | [SIGNAL] Minimum quality used in trimgalore and iVar trim | Default: 20 |
| main.signal_min_length | [SIGNAL] Minimum read length to retain after trimming | Default: 20 |
| main.artic_min_length | [ARTIC] Minimum read length for artic guppyplex | Default: 400 |
| main.artic_max_length | [ARTIC] Maximum read length for artic guppyplex | Default: 700 |
All [SIGNAL] file locations have been prefilled in the template input file. These files are downloaded and moved to the correct locations by the data download script.
accession, type, signal_scheme_bed, signal_primer_pairs_tsv, signal_amplicon_bed file locations, and artic_primer_version must be filled in on the template input file for the workflow to be run. For formatting help, check out the test input files in the same directory. Any inputs with default values defined to not need to be explicitly defined in the input file if no change to the default value is required.
For more information about SIGNAL parameters, see here. For more information about ARTIC parameters, see here.
- Example Illumina PE input file: inputs/from_fastq/illumina_pe.test_input.json
- Example Nanopore input file: inputs/from_fastq/nanopore.test_input.json
Primer schemes (the input to main.signal_scheme_bed) available in the data/primer_schemes directory are from the official artic-network github. The v3 scheme was altered such that it's 5th column was set to 60 for every row to conform with the BED specification (required by iVar).
Different schemes may be used for the Illumina PE workflow, e.g. schemes available in the SIGNAL github. The Nanopore workflow is currently only able to operate on ARTIC primer schemes (v1, v2, and v3).
Note that the download_fastqs task (the first task) relies on a reliable internet connection; connection to NCBI's SRA can sometimes be unstable. If the workflow fails at this step, try rerunning it at a later time when NCBI's servers may be under lighter load.
| Output identifier | Description | Notes |
|---|---|---|
| raw_reads | Fastq file(s) | 1 file for Nanopore runs; 2 for Illumina paired-end runs. |
| consensus_fa | Assembled genome consensus sequence | |
| vcf, vcf_index | High confidence variant calls and index | |
| bam | Read alignment to the SARS-CoV-2 reference | |
| full_output | Full output from each pipeline; differs between Nanopore and Illumina_PE pipelines | Contains additional quality metrics, trimmed alignments, etc. |
Ensure you have first filled out all input files appropriately.
From the root of the repository, run:
java -jar /path/to/cromwell.jar run workflows/from_fastq/main.wdl -i inputs/from_fastq/input_template.jsonOutput and execution files will be located in the cromwell-executions directory. When the workflow finishes successfully, it will output JSON (to stdout) specifying the full path to each output file.
Ensure you have first filled out all input files appropriately.
This command assumes you have miniwdl available on your command line. If miniwdl is not available, try installing using pip install miniwdl.
miniwdl run workflows/from_fastq/main.wdl -i inputs/from_fastq/input_template.jsonOutput and execution files will be located in a dated directory (e.g. named 20200704_073415_main). When the workflow finishes successfully, it will output JSON (to stdout) specifying the full path to each output file.