This is a Nextflow pipeline for running the FluViewer analysis tool and other custom modules to obtain consensus sequences, HA and NA subtypes, clade calls, and amino acid mutations for Influenza A WGS.
- Read trimming & QC: fastp
- Primer removal with cutadapt
- FASTQ quality reporting with FastQC
- Aggregate the reports with MultiQC
- Sequence analysis with BCCDC-PHL/FluViewer
- Extract HPAI motif (applies to H5 sequences only)
- Clade calls for H1, H3 and H5 influenza using Nextclade
- Amino acid SNP calls against a specified reference
- Genotype calls using GenoFLU against curated database
flowchart TD
fastq_input[FASTQ Input] --> fastp(fastp)
fastp -- trimmed_reads --> cutadapt(cutadapt)
cutadapt -- qc_stats --> multiqc(multiqc)
cutadapt -- primer_trimmed_reads --> fastqc(fastqc)
fastqc -- qc_stats --> multiqc
multiqc --> multiqc_report[MultiQC Report]
cutadapt -- primer_trimmed_reads --> normalize_reads(normalize_reads)
normalize_reads -- normalized_reads --> fluviewer(fluviewer)
fluviewer_db[FluViewer DB] --> fluviewer
fluviewer -- ha_consensus --> clade_calling(clade_calling)
clade_calling --> clade_calls[clade-calls]
fluviewer -- consensus_main --> snp_caling(snp_calling)
snp_calling --> snp_calls[snp-calls]
fluviewer -- consensus_main --> genoflu(genoflu)
fluviewer --> segment_coverage_plots[Segment Coverage Plots]
fluviewer --> consensus_sequence[Consensus Sequence]
fluviewer --> variants[Variants VCF]
genoflu --> genoflu_tsv[GenoFLU tsv]
Short read Illumina sequences, files ending in '.fastq.gz', '.fq.gz', '.fastq', '.fq'
Additional requirements for use:
- [optional] provide a database for use. If none is provided the tool should use the database bundled with FluViewer.
- NOTE: if you choose to use a custom database, you will need to make sure that the sequences and headers are formatted as FluViewer requires. See https://github.com/BCCDC-PHL/FluViewer for details.
- Package management is via Conda. In addition, note the
-profileand--cacheswitches, essential for proper operation of Conda on BCCDC systems.- NOTE: if you have problems when first running the pipeline, it can be due to problems resolving the Conda environment. If this is the case you can use mamba instead.
Optional arguments:
For a full list of optional arguments, see: https://github.com/BCCDC-PHL/FluViewer
| Argument | Description | Default Value |
|---|---|---|
--target_depth |
Depth to normalize coverage to, where sufficient depth is available in inputs. | 200 |
--min_depth |
Minimum read depth for base calling. | 20 |
--min_q |
Minimum PHRED score for base quality and mapping quality. | 20 |
--min_cov |
Minimum coverage of database reference sequence by contig, percentage. | 25 |
--min_ident |
Minimum nucleotide sequence identity between database reference sequence and contig (percentage) | 95 |
Example command:
nextflow run BCCDC-PHL/fluviewer-nf \
-r v0.3.0 \
-profile conda \
--cache ~/.conda/envs \
--fastq_input /path/to/your_fastqs \
--db /path/to/FluViewer_db.fa \
--outdir /path/to/output_dir
Outputs are written to the directory specified with the --outdir parameter. Below that are individual folders for each sample, containing the results of FluViewer, SNP calling, and clade calling.
<outdir>
├── <run_name>_fluviewer-nf_multiqc_report.html
├── <run_name>_fluviewer-nf_nextflow_report.html
├── <run_name>_fluviewer-nf_nextflow_timeline.html
└── <sample-id>
├── <sample-id>.fastp.html
├── <sample-id>.fastp.json
├── <sample-id>_<date-time>_provenance.yml
├── <sample-id>_HA_consensus.fa
├── <sample-id>_HPAI.tsv
├── <sample-id>_NA_consensus.fa
├── <sample-id>_alignment.bam
├── <sample-id>_alignment.bam.bai
├── <sample-id>_consensus_seqs.fa
├── <sample-id>_contigs_blast.tsv
├── <sample-id>_depth_of_cov.png
├── <sample-id>_fluviewer_provenance.yml
├── <sample-id>_genoflu.tsv
├── <sample-id>_mapping_refs.fa
├── <sample-id>_report.tsv
├── <sample-id>_variants.vcf
├── clade-calls
│ ├── <sample-id>_nextclade.aligned.fasta.gz
│ ├── <sample-id>_nextclade.csv
│ ├── <sample-id>_nextclade.json
│ ├── <sample-id>_nextclade.ndjson
│ ├── <sample-id>_nextclade.tsv
│ ├── <sample-id>_nextclade_HA1.translation.fasta.gz
│ ├── <sample-id>_nextclade_HA2.translation.fasta.gz
│ └── <sample-id>_nextclade_SigPep.translation.fasta.gz
├── fluviewer_logs
│ ├── 01_assemble_contigs
│ │ ├── spades_stderr.txt
│ │ └── spades_stdout.txt
│ ├── 02_blast_contigs
│ │ ├── blastn_contigs_stderr.txt
│ │ └── blastn_contigs_stdout.txt
│ ├── 03_scaffolding
│ │ ├── blastn_scaffolds_stderr.txt
│ │ └── blastn_scaffolds_stdout.txt
│ ├── 04_read_mapping
│ │ ├── bwa_index_stderr.txt
│ │ ├── bwa_index_stdout.txt
│ │ ├── bwa_mem_stderr.txt
│ │ ├── bwa_mem_stdout.txt
│ │ ├── samtools_index_stderr.txt
│ │ ├── samtools_index_stdout.txt
│ │ ├── samtools_view_stderr.txt
│ │ └── samtools_view_stdout.txt
│ ├── 05_variant_calling
│ │ ├── freebayes_stderr.txt
│ │ └── freebayes_stdout.txt
│ ├── 06_consensus_calling
│ │ ├── bcftools_consensus_stderr.txt
│ │ ├── bcftools_consensus_stdout.txt
│ │ ├── bcftools_index_stderr.txt
│ │ ├── bcftools_index_stdout.txt
│ │ ├── bcftools_view_stderr.txt
│ │ └── bcftools_view_stdout.txt
│ ├── 07_summary_reporting
│ │ ├── samtools_depth_stderr.txt
│ │ ├── samtools_depth_stdout.txt
│ │ ├── samtools_idxstats_stderr.txt
│ │ └── samtools_idxstats_stdout.txt
│ ├── fluviewer.log
│ ├── makeblastdb_contigs_stderr.txt
│ └── makeblastdb_contigs_stdout.txt
└── snp-calls
├── <sample-id>_HA_mutations.tsv
├── <sample-id>_M_mutations.tsv
├── <sample-id>_NA_mutations.tsv
├── <sample-id>_NP_mutations.tsv
├── <sample-id>_NS_mutations.tsv
├── <sample-id>_PA_mutations.tsv
├── <sample-id>_PB1_mutations.tsv
├── <sample-id>_PB2_mutations.tsv
└── pairwise
Output for each sample includes:
| File | Description |
|---|---|
*_report.tsv |
contains assembly and reconstruction metrics for all segments |
*_consensus_seqs.fa |
multifasta containing the consesus sequences for each segment |
*_HA_consensus.fa |
consensus sequnce for the HA segment (extracted from the multifasta produced by FluViewer) |
*_NA_consensus.fa |
consensus sequnce for the NA segment (extracted from the multifasta produced by FluViewer) |
*_depth_of_cov.png |
line plots describing mapping coverage across all 8 flu segments |
*_variants.vcf |
list of mutations in variant call format (VCF) called relative to the reference sequence |
snp-calls/*_mutations.tsv |
tabular list of amino acid SNP mutations relative to the reference |
genoflu/*_stats.tsv |
tabular list of genotype call information for the sample's consensus sequences |
Output for each run includes:
| File | Description |
|---|---|
multiqc_report.html |
MultiQC report summarizing the fastp results for the all the samples in the run |
For each pipeline invocation, each sample will produce a provenance.yml file with the following contents. Note the below is a contrived example.
- pipeline_name: BCCDC-PHL/fluviewer-nf
pipeline_version: 0.3.0
nextflow_session_id: 59fdd919-fc28-4af5-99e0-60355b11807c
nextflow_run_name: hopeful_bhaskara
timestamp_analysis_start: 2024-07-15T16:30:11.150887-07:00
- input_filename: sample-01_R1.fastq.gz
file_type: fastq_input
sha256: 2c1d5b310a5ca11cc2b1665c094a064fc3aa597e06f392985dac639bd2ab4d81
- input_filename: sample-01_R2.fastq.gz
file_type: fastq_input
sha256: 73745eed4badc3594cdd8554e90c75ae4b9b4599ca255064415ded433e598749
- process_name: fastp
tools:
- tool_name: fastp
tool_version: 0.23.2
- process_name: cutadapt
tools:
- tool_name: cutadapt
tool_version: 4.4
- process_name: normalize_depth
tools:
- tool_name: bbnorm
tool_version: 39.01
- process_name: fluviewer
tools:
- tool_name: fluviewer
tool_version: 0.1.11-6
databases:
- database_name: FluViewer_db.fa
database_path: /path/to/FluViewer_db.fa
database_sha256: c9ba1af0a637671d86a14aceac3cbfde309ae9a5bd613d75c87ba2ff390b4c48
process_name: nextclade
tools:
- tool_name: nextclade
tool_version: 3.8.1
subcommand: run
- process_name: snp_caling
tools:
- tool_name: blastx
tool_version: 2.15.0+
databases:
- database_name: blastx_subtype_db.fasta
- process_name: genoflu
tools:
- tool_name: genoflu
tool_version: 1.03