This document describes the output produced by the pipeline.

The directories listed below will be created in the results directory after the pipeline has finished. All paths are relative to the top-level results directory.

Pipeline overview

The pipeline is built using Nextflow and processes data using the following steps:

Reference Indexing

Output files

  • bwa/index.
    • *.amb
    • *.ann
    • *.bwt
    • *.pac
    • *.sa

These files are generally not required except for in the mapping step

Read Trimming

Output files

  • fastp/
    • *.html html reports of the trimming process that can be opened in any modern web browser
    • *.json trimming report metrics in JSON computer readable formats

Read Subsampling

Output files

  • rasusa/
    • *.fastq.gz subsamples fastq files

Read Mapping

By default there are no output files since sorted bam files are produced in the next step

Sort Bam Files

Output files

  • samtools/
    • *.bam sorted bam files
    • *.bam.bai bam file index
    • *.bam.flagstat bam file metrics
    • *.bam.idxstats bam file metrics
    • *.bam.stats bam file metrics

Call and Filter Variants

Output files

  • variants/
    • *.vcf.gz filtered vcf files containing variants

Convert Filtered VCF to Pseudogenome

Output files

  • pseudogenomes/
    • *.fas pseudogenome with a base at each position of the reference sequence

Create Alignment from Pseudogenomes

Only those pseudogenome fasta files that have a non-ACGT fraction less than the threshold specified will be included in the aligned_pseudogenomes.fas file. Those failing this will be reported in the low_quality_pseudogenomes.tsv file.

Output files

  • pseudogenomes/
    • aligned_pseudogenomes.fas alignment of all sample pseudogenomes and the reference sequence
    • low_quality_pseudogenomes.tsv a tab separated file of the samples that failed the non-ACGT base threshold

Remove Recombination

The file used for downstream tree building is aligned_pseudogenomes.filtered_polymorphic_sites.fasta. The other files are described in the gubbins documentation

Output files

  • gubbins/
    • aligned_pseudogenomes.branch_base_reconstruction.embl
    • aligned_pseudogenomes.filtered_polymorphic_sites.fasta
    • aligned_pseudogenomes.filtered_polymorphic_sites.phylip
    • aligned_pseudogenomes.final_tree.tre
    • aligned_pseudogenomes.node_labelled.final_tree.tre
    • aligned_pseudogenomes.per_branch_statistics.csv
    • aligned_pseudogenomes.recombination_predictions.embl
    • aligned_pseudogenomes.recombination_predictions.gff
    • aligned_pseudogenomes.summary_of_snp_distribution.vcf

Remove Non-informative Positions

Output files

  • snpsites/
    • constant.sites.txt A file with the number of constant sites for each base
    • filtered_alignment.fas Alignment with only informative positions (those positions that have at least one alternative variant base)


Output files

  • rapidnj/
    • rapidnj_phylogeny.tre A newick tree built with RapidNJ


Output files

  • fasttree/
    • fasttree_phylogeny.tre A newick tree built with FastTree


Output files

  • iqtree/
    • *.treefile A ML tree built with IQ-TREE with support values for branches based on bootstrapping


Output files

  • iqtree/
    • output.raxml.bestTree A ML tree built with RAxML-NG selected as the best after running ML
    • A ML tree built with RAxML-NG with support values for branches based on bootstrapping


Various quality statistics are compiled from the previous outputs using the MultiQC software:

Overall Statistics

A compilation of statistics about read content, mapping and variants General Statistics

FastP Statistics

Statistics gathered when trimming reads FastP Statistics

Mapping Statistics

Statistics gathered when mapping reads Mapping Statistics

Varinat Statistics

Statistics gathered when calling variants after filtering Variant Statistics

Pipeline information

Output files
  • pipeline_info/
    • Reports generated by Nextflow: execution_report.html, execution_timeline.html, execution_trace.txt and
    • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and software_versions.csv.
    • Reformatted samplesheet files used as input to the pipeline: samplesheet.valid.csv.

Nextflow provides excellent functionality for generating various reports relevant to the running and execution of the pipeline. This will allow you to troubleshoot errors with the running of the pipeline, and also provide you with other information such as launch commands, run times and resource usage.