fluviewer-nf

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.

Analyses

• 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]

Input

Short read Illumina sequences, files ending in '.fastq.gz', '.fq.gz', '.fastq', '.fq'

Usage

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 -profile and --cache switches, 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

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

Output

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:

Output for each run includes:

Provenance files

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