dragonflye-nf

A generic pipeline for creating long-read assemblies. Supports both long (Oxford Nanopore) reads, or hybrid assemblies with both short and long reads. Optionally annotate genes. Collects quality info on both incoming and outgoing datasets.

Analyses

• Read trimming & QC: fastp and filtlong
• Genome Assembly: dragonflye (long reads, or hybrid)
• Gene Annotation: prokka or bakta
• Assembly QC: quast, bandage

Usage

By default, dragonflye will be used for assembly of long reads, and no gene annotation will be run:

nextflow run BCCDC-PHL/dragonflye-nf \
  --long_only \
  --fastq_input_long <long-read fastq input directory> \
  --outdir <output directory>

...or a hybrid assembly can be generated by supplying short reads:

nextflow run BCCDC-PHL/dragonflye-nf \
  --hybrid \
  --fastq_input <short-read fastq input directory> \
  --fastq_input_long <long-read fastq input directory> \
  --outdir <output directory>

Prokka and/or bakta can be used with the --prokka and --bakta flags:

nextflow run BCCDC-PHL/dragonflye-nf \
  --long_only \
  --fastq_input_long <long-read fastq input directory> \
  --prokka \
  --bakta \
  --outdir <output directory>

The pipeline also supports a 'samplesheet input' mode. Pass a samplesheet.csv file with the headers ID, R1, R2,LONG:

nextflow run BCCDC-PHL/dragonflye-nf \
  --samplesheet_input <samplesheet.csv> \
  --outdir <output directory>

Eg:

ID,R1,R2,LONG
sample-01,/path/to/sample-01_R1.fastq.gz,/path/to/sample-01_R2.fastq.gz,/path/to/sample-01_RL.fastq.gz
sample-02,/path/to/sample-02_R1.fastq.gz,/path/to/sample-02_R2.fastq.gz,/path/to/sample-02_RL.fastq.gz
sample-03,/path/to/sample-03_R1.fastq.gz,/path/to/sample-03_R2.fastq.gz,/path/to/sample-03_RL.fastq.gz

By default, dragonflye will tag circularized contigs with a circular=Y annotation in the fasta header, and circular=N for linear contigs. For example:

>contig00001 len=5202987 cov=191.0 origname=contig_1_polypolish polish=racon:1 round(s);polypolish:short_reads,1 round(s); sw=dragonflye-flye/1.1.0 date=20230912 circular=Y
>contig00002 len=3964 cov=155.0 origname=contig_4_polypolish polish=racon:1 round(s);polypolish:short_reads,1 round(s); sw=dragonflye-flye/1.1.0 date=20230912 circular=N
...

In contrast, unicycler adds a circular=true tag to circularized contigs and no circularization tag to linear contigs. For example:

>1 length=5202987 depth=191.0x circular=true
>2 length=3964 depth=155.0

Both this pipeline and our BCCDC-PHL/routine-assembly edit the fasta header to add the sample ID to the front. In addition, this pipeline accepts a --use_unicycler_circularization_tag flag that will convert circular=Y to circular=true and will remove circular=N.

nextflow run BCCDC-PHL/dragonflye-nf \
  --hybrid \
  --use_unicycler_circularization_tag \
  --fastq_input <short-read fastq input directory> \
  --fastq_input_long <long-read fastq input directory> \
  --outdir <output directory>

The --meta flag can be passed through to flye by adding the --flye_meta flag. The --meta flag is intended for use with metagenome assemblies, but may be helpful in other scenarios such as plasmid assembly:

nextflow run BCCDC-PHL/dragonflye-nf \
  --hybrid \
  --flye_meta \
  --fastq_input <short-read fastq input directory> \
  --fastq_input_long <long-read fastq input directory> \
  --outdir <output directory>

Output

An output directory will be created for each sample under the directory provided with the --outdir flag. The directory will be named by sample ID, inferred from the fastq files (all characters before the first underscore in the fastq filenames), or the ID field of the samplesheet, if one is used.

If we have sample-01_R{1,2}.fastq.gz, in our --fastq_input directory, the output directory will be:

sample-01
├── sample-01_20211125165316_provenance.yml
├── sample-01_fastp.csv
├── sample-01_fastp.json
├── sample-01_dragonflye_short.fa
├── sample-01_dragonflye_short.log
└── sample-01_dragonflye_short_quast.csv

Including the tool name suffixes on output files allows re-analysis of the same sample with multiple tools without conflicting output filenames:

sample-01
├── sample-01_20211125165316_provenance.yml
├── sample-01_20211128122118_provenance.yml
├── sample-01_fastp.csv
├── sample-01_fastp.json
├── sample-01_dragonflye_hybrid_bakta.gbk
├── sample-01_dragonflye_hybrid_bakta.gff
├── sample-01_dragonflye_hybrid_bakta.json
├── sample-01_dragonflye_hybrid_bakta.log
├── sample-01_dragonflye_hybrid_bandage.png
├── sample-01_dragonflye_hybrid_prokka.gbk
├── sample-01_dragonflye_hybrid_prokka.gff
├── sample-01_dragonflye_hybrid_quast.csv
├── sample-01_dragonflye_hybrid_quast.csv
├── sample-01_dragonflye_hybrid.fa
├── sample-01_dragonflye_hybrid.gfa
├── sample-01_dragonflye_hybrid.log
├── sample-01_dragonflye_short_bakta.gbk
├── sample-01_dragonflye_short_bakta.gff
├── sample-01_dragonflye_short_bakta.json
├── sample-01_dragonflye_short_bakta.log
├── sample-01_dragonflye_short_bandage.png
├── sample-01_dragonflye_short_prokka.gbk
├── sample-01_dragonflye_short_prokka.gff
├── sample-01_dragonflye_short_quast.csv
├── sample-01_dragonflye_short_quast.csv
├── sample-01_dragonflye_short.fa
├── sample-01_dragonflye_short.gfa
└── sample-01_dragonflye_short.log

If the --versioned_outdir flag is used, then a sub-directory will be created below each sample, named with the pipeline name and minor version:

sample-01
    └── dragonflye-nf-v0.4-output
        ├── sample-01_20220216172238_provenance.yml
        ├── sample-01_fastp.csv
        ├── sample-01_fastp.json
        ├── sample-01_dragonflye_short.fa
        ├── sample-01_dragonflye_short.log
        ├── sample-01_dragonflye_short_prokka.gbk
        ├── sample-01_dragonflye_short_prokka.gff
        └── sample-01_dragonflye_short_quast.csv

This is provided as a way of combining outputs of several different pipelines or re-analysis with future versions of this pipeline:

sample-01
    └── dragonflye-nf-v0.4-output
    │   ├── sample-01_20220216172238_provenance.yml
    │   ├── sample-01_fastp.csv
    │   ├── sample-01_fastp.json
    │   ├── sample-01_dragonflye_short.fa
    │   ├── sample-01_dragonflye_short.log
    │   ├── sample-01_dragonflye_short_prokka.gbk
    │   ├── sample-01_dragonflye_short_prokka.gff
    │   └── sample-01_dragonflye_short_quast.csv
    └── dragonflye-nf-v0.5-output
        ├── sample-01_20220612091224_provenance.yml
        ├── sample-01_fastp.csv
        ├── sample-01_fastp.json
        ├── sample-01_dragonflye_short.fa
        ├── sample-01_dragonflye_short.log
        ├── sample-01_dragonflye_short_prokka.gbk
        ├── sample-01_dragonflye_short_prokka.gff
        └── sample-01_dragonflye_short_quast.csv

Provenance files

For each pipeline invocation, each sample will produce a provenance.yml file with the following contents:

- pipeline_name: BCCDC-PHL/dragonflye-nf
  pipeline_version: 0.4.0
- timestamp_analysis_start: 2022-08-16T13:22:11.553143
- input_filename: sample-01_R1.fastq.gz
  sha256: 4ac3055ac5f03114a005aff033e7018ea98486cbebdae669880e3f0511ed21bb
  file_type: fastq-input
- input_filename: sample-01_R2.fastq.gz
  sha256: 8db388f56a51920752319c67b5308c7e99f2a566ca83311037a425f8d6bb1ecc
  file_type: fastq-input
- process_name: fastp
  tools:
    - tool_name: fastp
      tool_version: 0.23.1
- process_name: dragonflye
  tools:
    - tool_name: dragonflye
      tool_version: 1.1.0
- process_name: prokka
  tools:
    - tool_name: prokka
      tool_version: 1.14.5
      parameters:
        - parameter: --compliant
          value: null
- process_name: quast
  tools:
    - tool_name: quast
      tool_version: 5.0.2
      parameters:
        - parameter: --space-efficient
          value: null
        - parameter: --fast
          value: null

The filename of the provenance file includes a timestamp with format YYYYMMDDHHMMSS to ensure that re-analysis of the same sample will create a unique provenance.yml file.