A generic pipeline for creating routine draft assemblies. Supports both short (illumina) and 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.
- Read trimming & QC: fastp and filtlong
- Genome Assembly: unicycler (short reads, long reads, or hybrid)
- Gene Annotation: prokka or bakta
- Assembly QC: quast, bandage
flowchart TD
short_reads --> fastp(fastp)
long_reads --> filtlong(filtlong)
filtlong -- filtered_reads --> unicycler(unicycler)
fastp -- trimmed_reads --> unicycler
unicycler --> assembly["assembly.fa"]
unicycler --> prokka(prokka)
prokka --> prokka_genes["prokka_genes.gff"]
unicycler --> bakta(bakta)
bakta --> bakta_genes["bakta_genes.gff"]
unicycler --> quast(quast)
unicycler --> bandage(bandage)
bandage --> bandage_diagram["bandage_diagram.png"]
quast --> assembly_qc["assembly_qc.csv"]
By default, unicycler will be used for assembly of short reads, and no gene annotation will be run:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input <fastq input directory> \
--outdir <output directory>
Prokka and/or bakta can be used with the --prokka and --bakta flags:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input <fastq input directory> \
--prokka \
--bakta \
--outdir <output directory>
Any combination of short/hybrid/long_only and prokka/bakta is supported: Short reads with bakta:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input <fastq input directory> \
--bakta \
--outdir <output directory>
...long reads with prokka:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input_long <fastq input directory> \
--long_only \
--prokka \
--outdir <output directory>
...hybrid with both:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input <fastq input directory> \
--fastq_input_long <fastq input directory> \
--hybrid \
--prokka \
--bakta \
--outdir <output directory>
The pipeline also supports a 'samplesheet input' mode. Pass a samplesheet.csv file with the headers ID, R1, R2:
nextflow run BCCDC-PHL/routine-assembly-nf \
--samplesheet_input <samplesheet.csv> \
--outdir <output directory>
Eg:
ID,R1,R2
sample-01,/path/to/sample-01_R1.fastq.gz,/path/to/sample-01_R2.fastq.gz
sample-02,/path/to/sample-02_R1.fastq.gz,/path/to/sample-02_R2.fastq.gz
sample-03,/path/to/sample-03_R1.fastq.gz,/path/to/sample-03_R2.fastq.gz
If long (Oxford Nanopore) and short (illumina) reads are both available, hybrid assemblies can be performed with unicycler. Note that shovill does not support hybrid assemblies.
Add the --hybrid flag to perform hybrid assembly, and supply long reads using the --long_reads flag. Sample IDs for both long and short reads are taken from all characters of the
fastq files up to the first underscore _. Sample IDs for short and long reads must match in order for the pipeline to match them up for hybrid assembly.
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input <fastq input directory> \
--fastq_input_long <long reads input directory> \
--hybrid \
--outdir <output directory>
Hybrid assembly mode is compatible with samplesheet input mode. When using a samplesheet for hybrid assemblies, an additional field with header LONG is required.
Note that in this mode, because the sample IDs are explicitly provided in the samplesheet, it isn't strictly necessary that the short and long read filenames have matching sample IDs
in their filenames (though it's still probably good practice to do so).
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_L.fastq.gz
sample-02,/path/to/sample-02_R1.fastq.gz,/path/to/sample-02_R2.fastq.gz,/path/to/sample-02_L.fastq.gz
sample-03,/path/to/sample-03_R1.fastq.gz,/path/to/sample-03_R2.fastq.gz,/path/to/sample-03_L.fastq.gz
All samples in the samplesheet should have both short and long reads when running in hybrid assembly mode.
Run the pipeline as follows:
nextflow run BCCDC-PHL/routine-assembly \
--samplesheet /path/to/samplesheet.csv \
--hybrid \
--outdir <output directory>
If only long (Oxford Nanopore) reads are available, a long-read-only assembly mode is supported. Use the --long_only flag, as follows:
nextflow run BCCDC-PHL/routine-assembly \
--fastq_input_long <long reads input directory> \
--long_only \
--outdir <output directory>
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_unicycler_short.fa
├── sample-01_unicycler_short.log
└── sample-01_unicycler_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_unicycler_hybrid_bakta.gbk
├── sample-01_unicycler_hybrid_bakta.gff
├── sample-01_unicycler_hybrid_bakta.json
├── sample-01_unicycler_hybrid_bakta.log
├── sample-01_unicycler_hybrid_bandage.png
├── sample-01_unicycler_hybrid_prokka.gbk
├── sample-01_unicycler_hybrid_prokka.gff
├── sample-01_unicycler_hybrid_quast.csv
├── sample-01_unicycler_hybrid_quast.csv
├── sample-01_unicycler_hybrid.fa
├── sample-01_unicycler_hybrid.gfa
├── sample-01_unicycler_hybrid.log
├── sample-01_unicycler_short_bakta.gbk
├── sample-01_unicycler_short_bakta.gff
├── sample-01_unicycler_short_bakta.json
├── sample-01_unicycler_short_bakta.log
├── sample-01_unicycler_short_bandage.png
├── sample-01_unicycler_short_prokka.gbk
├── sample-01_unicycler_short_prokka.gff
├── sample-01_unicycler_short_quast.csv
├── sample-01_unicycler_short_quast.csv
├── sample-01_unicycler_short.fa
├── sample-01_unicycler_short.gfa
└── sample-01_unicycler_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
└── routine-assembly-v0.4-output
├── sample-01_20220216172238_provenance.yml
├── sample-01_fastp.csv
├── sample-01_fastp.json
├── sample-01_unicycler_short.fa
├── sample-01_unicycler_short.log
├── sample-01_unicycler_short_prokka.gbk
├── sample-01_unicycler_short_prokka.gff
└── sample-01_unicycler_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
└── routine-assembly-v0.4-output
│ ├── sample-01_20220216172238_provenance.yml
│ ├── sample-01_fastp.csv
│ ├── sample-01_fastp.json
│ ├── sample-01_unicycler_short.fa
│ ├── sample-01_unicycler_short.log
│ ├── sample-01_unicycler_short_prokka.gbk
│ ├── sample-01_unicycler_short_prokka.gff
│ └── sample-01_unicycler_short_quast.csv
└── routine-assembly-v0.5-output
├── sample-01_20220612091224_provenance.yml
├── sample-01_fastp.csv
├── sample-01_fastp.json
├── sample-01_unicycler_short.fa
├── sample-01_unicycler_short.log
├── sample-01_unicycler_short_prokka.gbk
├── sample-01_unicycler_short_prokka.gff
└── sample-01_unicycler_short_quast.csv
For each pipeline invocation, each sample will produce a provenance.yml file with the following contents:
- pipeline_name: BCCDC-PHL/routine-assembly
pipeline_version: 0.4.5
nextflow_session_id: aad504aa-848f-4e85-8a9c-2aa43901d1b4
nextflow_run_name: pedantic_carson
timestamp_analysis_start: 2024-04-25T10:37:19.351714-07:00
- 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: unicycler
tools:
- tool_name: unicycler
tool_version: 0.4.8
- 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: nullThe 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.