plasmid-screen

Usage

This pipeline is designed to take either raw reads alone, or assemblies plus raw reads as input. If only reads are provided, they will be assembled with unicycler.

nextflow run BCCDC-PHL/plasmid-screen \
  --fastq_input </path/to/fastqs> \
  --mob_db </path/to/mob-suite-db> \
  --outdir </path/to/outdir> 

If assemblies are already available, they can be provided by adding the --pre_assembled flag, and supplying the assemblies to the --assembly_input flag.

nextflow run BCCDC-PHL/plasmid-screen \
  --pre_assembled \
  --assembly_input </path/to/assemblies> \
  --fastq_input </path/to/fastqs> \
  --mob_db </path/to/mob-suite-db> \
  --outdir </path/to/outdir> 

Alternatively, a 'samplesheet.csv' file may be provided with fields ID, R1, R2:

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
...

nextflow run BCCDC-PHL/plasmid-screen \
  --samplesheet_input </path/to/samplesheet.csv> \
  --mob_db </path/to/mob-suite-db> \
  --outdir </path/to/outdir> 

...or if assemblies are available, the samplesheet.csv file may also include the field ASSEMBLY:

ID,R1,R2,ASSEMBLY
sample-01,/path/to/sample-01_R1.fastq.gz,/path/to/sample-01_R2.fastq.gz,/path/to/sample-01.fa
sample-02,/path/to/sample-02_R1.fastq.gz,/path/to/sample-02_R2.fastq.gz,/path/to/sample-01.fa
...

nextflow run BCCDC-PHL/plasmid-screen \
  --pre_assembled \
  --samplesheet_input </path/to/samplesheet.csv> \
  --mob_db </path/to/mob-suite-db> \
  --outdir </path/to/outdir> 

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
    └── plasmid-screen-v0.2-output
        ├── sample-01_abricate.tsv
        ├── sample-01_chromosome.fasta
        ├── sample-01_fastp.csv
        ├── sample-01_mash_screen.tsv
        ├── ...
        ├── sample-01_quast.csv
        └── NC_019152.1.fa

Outputs

The main output of the pipeline is the 'Resistance gene report', which summarizes where the resistance gene was located (contig and position), the quality of the resitance gene match (% identity and % coverage) and a characterization of the plasmid reconstruction. The report includes the following fields:

sample_id
assembly_file
resistance_gene_contig_id
resistance_gene_contig_size
resistance_gene_id
resistance_gene_contig_position_start
resistance_gene_contig_position_end
percent_resistance_gene_coverage
percent_resistance_gene_identity
num_contigs_in_plasmid_reconstruction
plasmid_reconstruction_size
replicon_types
mob_suite_primary_cluster_id
mob_suite_secondary_cluster_id
mash_nearest_neighbor
mash_neighbor_distance
alignment_ref_plasmid
depth_coverage_threshold
percent_ref_plasmid_coverage_above_depth_threshold
num_snps_vs_ref_plasmid

Additional Output Files

For each sample, the following output files are created:

sample-01/
├── sample-01_20211207163723_provenance.yml
├── sample-01_abricate.tsv
├── sample-01_chromosome.fasta
├── sample-01_fastp.csv
├── sample-01_mash_screen.tsv
├── sample-01_mobtyper_contig_report.tsv
├── sample-01_mobtyper_plasmid_report.tsv
├── sample-01_resistance_gene_report.tsv
├── sample-01_NC_019152.1.snps.vcf
├── sample-01_NC_019152.1.sorted.bam
├── sample-01_NC_019152.1.sorted.bam.bai
├── sample-01_plasmid_AA023.fasta
├── sample-01_plasmid_AA026.fasta
├── sample-01_quast.csv
└── NC_019152.1.fa

filename suffix	Generated by	Description
_abricate.tsv	abricate	All resistance genes found in the entire assembly
_chromosome.fasta	mob_recon	The set of contigs determined by mob_recon to belong to the chromosome (non-plasmid)
_plasmid_<cluster_id>_.fasta	mob_recon	Plasmid reconstructions. Groups of contigs that were determined to be part of the same plasmid
_fastp.csv	fastp	Read QC info
_quast.csv	quast	Assembly QC info
_mash_screen.tsv	mash	Containment of reference plasmids in reads
_mobtyper_contig_report.csv	mob_typer	MOB Typer results for all contigs in the assembly (both chromosome and plasmid)
_mobtyper_plasmid_report.csv	mob_typer	MOB Typer results for all plasmid reconstructions (including those that do not have resistance genes
_<plasmid_id>.sorted.bam{.bai}	bwa	Alignment of reads against a reference plasmid
.snps.vcf	freebayes	SNPs found in alignment of reads against a reference plasmid
<plasmid_id>.fa	seqkit	Reference plasmid used for alignments

Provenance

Each analysis will create a provenance.yml file for each sample. The filename of the provenance.yml file includes a timestamp with format YYYYMMDDHHMMSS to ensure that a unique file will be produced if a sample is re-analyzed and outputs are stored to the same directory.

Example provenance output:

- process_name: mob_recon
  tool_name: mob_recon
  tool_version: 3.0.3
  parameters:
  - parameter: database_directory
    value: /path/to/mob_db
  - parameter: filter_db
    value: /path/to/mob_filter_db
  - parameter: min_con_cov
    value: 95
- process_name: abricate
  tool_name: abricate
  tool_version: 1.0.1
  parameters:
  - parameter: db
    value: ncbi
- process_name: trim_reads
  tool_name: fastp
  tool_version: 0.22.0
  parameters:
  - parameter: cut_tail
    value: true
- input_filename: sample-01_R1.fastq.gz
  input_path: /path/to/sample-01_R1_001.fastq.gz
  sha256: b0534592d61321243897e842a9ea655d396d4496cbf6d926b6c6fea8e06aa98d
- input_filename: sample-01_R2.fastq.gz
  input_path: /path/to/sample-01_R2_001.fastq.gz
  sha256: cc66309103da91e337143eb649196d84ed3ebe2ff08a45b197cd4151d137a167
- input_filename: sample-01.fa
  input_path: /path/to/sample-01.fa
  sha256: 6fffb542711ee301ef1185a403a74fed36c066872e3fbfb7aa5c81464243bd00
- process_name: align_reads_to_reference_plasmid
  process_tags:
    ref_plasmid_id: NC_019152.1
    resistance_gene: blaKPC-3
  tool_name: bwa
  subcommand: mem
  tool_version: 0.7.17-r1188
  parameters:
  - parameter: alignment_algorithm
    value: mem
- process_name: align_reads_to_reference_plasmid
  process_tags:
    ref_plasmid_id: NC_019152.1
    resistance_gene: blaKPC-3
  tool_name: samtools
  subcommand: view
  tool_version: 1.13
  parameters:
  - parameter: exclude_flags
    value: 1540
- process_name: call_snps
  process_tags:
    ref_plasmid_id: NC_019152.1
    resistance_gene: blaKPC-3
  tool_name: freebayes
  tool_version: 1.3.5
  parameters:
  - parameter: ploidy
    value: 1
  - parameter: min_base_quality
    value: 20
  - parameter: min_mapping_quality
    value: 60
  - parameter: min_coverage
    value: 10
  - parameter: min_alternate_fraction
    value: 0.8
  - parameter: min_repeat_entropy
    value: 1.0
- process_name: call_snps
  process_tags:
    ref_plasmid_id: NC_019152.1
    resistance_gene: blaKPC-3
  tool_name: bcftools
  subcommand: view
  tool_version: 1.12
  parameters:
  - parameter: include
    value: INFO/TYPE=snp
- process_name: quast
  tool_name: quast
  tool_version: 5.0.2
- pipeline_name: BCCDC-PHL/plasmid-screen
  pipeline_version: 0.1.0
- timestamp_analysis_start: 2021-12-06T16:12:31.252055