Dryad is a NextFlow pipeline to construct reference free core-genome or SNP phylogenetic trees for examining prokaryote relatedness in outbreaks. Dryad will performs both a reference free core-genome analysis based off of the approach outlined by Oakeson et. al and/or a SNP analysis using the CFSAN-SNP pipeline.
Usage
Workflow outline
Core-genome
SNP
Quality assessment
Output
The pipeline is designed to start from raw Illumina short reads. All reads must be in the same directory. Then start the pipeline using:
nextflow wslh-bio/dryad -r <version> --reads [path-to-reads]
to run the SNP pipeline include the --snp_reference parameter:
nextflow wslh-bio/dryad -r <version> --reads [path-to-reads] --snp_reference [path-to-reference-fasta]
You can also test the pipeline with example data using --test Note: This requires NextFlow version 21.07.0-edge or greater:
nextflow dryad.nf --test
Read trimming and cleaning is performed using BBtools v38.76 to trim reads of low quality bases and remove PhiX contamination. After processing, the reads are used by each pipeline as needed.
Note: Both pipelines can be run automatically in parallel by supplying the snp_reference parameter.
The core genome pipeline takes the trimmed and cleaned reads and infers a phylogenetic tree that can be used for inferring outbreak relatedness. This pipeline is based loosely off of the pipeline described here by Oakeson et. al.
Species is predicted from the cleaned reads, and assembly quality is evaluated.
The core genome pipeline uses the following applications and pipelines:
Shovill v1.0.4 Shovill is a genome assembly pipeline that uses SPAdes, but it alters certain steps to get similar results in less time.
Prokka v1.14.5 Prokka is a whole genome annotation tool used to annotate the coding regions of the assembly.
Roary v3.12.0 Roary takes the annotated genomes and constructs a core gene alignment.
IQ-Tree v1.6.7 IQ-Tree uses the core gene alignment and infers a maximum likelihood phylogenetic tree bootstrapped 1000 times.
QUAST v5.0.2 QUAST evaluates genome assembly quality.
Kraken2 v2.0.8 Kraken uses the cleaned reads to predict species and detect contamination.
The SNP pipeline takes the trimmed and cleaned reads and infers a phylogenetic tree that can be used for inferring outbreak relatedness. The pipeline requires the path to the raw reads (mentioned above) and a reference genome in fasta file format.
The SNP pipeline uses the following applications and pipelines:
IQ-Tree v1.6.7 IQ-Tree uses an alignment of the SNP sites to create a maximum likelihood phylogenetic tree bootstrapped 1000 times.
FastQC v0.11.8 FastQC is used assess the quality of the raw and cleaned reads.
QUAST v5.0.2 QUAST assesses the quality of the genome assemblies.
Samtools v1.10 calculates the number and depth of cleaned reads mapped to their assemblies and the reference genome. BWA v0.7.17-r1188 is used for mapping reads.
MultiQC v1.8 summarizes the results of FastQC, Prokka, Samtools Stats and Kraken.
dryad_results
├── annotated
│ ├── *.gff
│ └── *.prokka.stats.txt
├── assembled
│ └── *.contigs.fa
├── core_gene_alignment.aln
├── core_genome_statistics.txt
├── core_genome.tree
├── dryad_report.csv
├── assembled
├── fastqc
│ ├── fastqc_summary.txt
│ ├── *.html
│ └── zips
│ └── *.zip
├── kraken
│ ├── kraken_results.tsv
│ └── *.kraken2.txt
├── mapping
│ ├── bams
│ │ ├── *.assembly.bam
│ │ ├── *.assembly.bai
│ │ ├── *.reference.bam
│ │ └── *.reference.bai
│ ├── coverage_stats.tsv
│ ├── depth
│ │ ├── *.assembly.depth.tsv
│ │ └── *.reference.depth.tsv
│ ├── mapping_stats.tsv
│ ├── sams
│ │ ├── *.assembly.sam
│ │ └── *.reference.sam
│ └── stats
│ ├── *.assembly.stats.txt
│ └── *.reference.stats.txt
├── multiqc_report.html
├── quast
│ ├── quast_results.tsv
│ └── *.quast.tsv
├── snp_distance_matrix.tsv
├── snpma.fasta
├── snp.tree
└── trimming
├── bbduk_results.tsv
├── reads
│ └── *_clean_*
└── stats
└── *.trim.txt
*.gff - Gene annotations predicted by Prokka
*.prokka.stats.txt - Prokka log files
*.contigs.fa - Shovill assemblies
core_gene_alignment.aln - Core-genome alignment
core_genome_statistics.txt - Text file with the number of genes in the core and accessory genomes
core_genome.tree - ML tree inferred from core-genome alignment
dryad_report.csv - Summary table of each step in dryad
fastqc_summary.txt - Summary table of FastQC results
*.html - HTML files of FastQC results
*.zip - FastQC results, compressed
kraken_results.tsv - Summary table of Kraken results
*.kraken2.txt - Report of Kraken results for each sample
*.assembly.bam - Alignments to an assembly BAM format
*.assembly.bai - Index file of alignments to an assembly
*.reference.bam - Alignments to the reference sequence BAM format
*.reference.bai - Index file of alignments to the reference sequence
coverage_stats.tsv - Summary table of mean and median coverage calculated with Samtools depth
*.assembly.depth.tsv - Raw Samtools depth output for reads mapped to an assembly
*.reference.depth.tsv - Raw Samtools depth output for reads mapped to the reference sequence
mapping_stats.tsv - Summary table of statistics
*.assembly.sam - Alignments to an assembly SAM format
*.reference.sam - Alignments to the reference sequence SAM format
*.assembly.stats.txt - Output of Samtools stats for reads mapped to an assembly
*.reference.stats.txt - Output of Samtools stats for reads mapped to the reference sequence
multiqc_report.html - HTML report generated by MultiQC
quast_results.tsv - Summary table of QUAST results
*.quast.tsv - QUAST results for each sample
snp_distance_matrix.tsv - SNP distance matrix
snpma.fasta - SNP alignment
snp.tree - SNP tree
bbduk_results.tsv - Summary table of trimming with BBDuk
*_clean_* - Trimmed and cleaned reads
*.trim.txt - Trimming results from BBduk each sample
Kelsey Florek, WSLH Senior Genomics and Data Scientist
Abigail Shockey, WSLH Bioinformatics and Data Scientist