Introduction Usage Parameters Output Releases

Introduction

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

In order to map the reads to the reference sequence it indexed.

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

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

Read Trimming

The fastp software is used to trim the fastq input files.

Output files
  • fastp/
    • *.html html reports of the trimming process that can be opened in any modern web browser. See here for an example
    • *.json trimming report metrics in JSON computer readable formats. See here for an example

Read Subsampling

The rasusa software is used to subsample reads to a depth cutoff of a default of 100 (unless the --subsampling_off flag is set)

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

Read Mapping

By default there are the bam files created are not saved since sorted bam files are produced in the next step

Sort Bam Files

After mapping the bam files are sorted and statistics calculated.

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

The bcftools software is used to call and filter variants found within the bam files.

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

Convert Filtered VCF to Pseudogenome

The filtered vcf files are converted to a 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

Before building trees, non-informative constant sites are removed from the alignment using snp-sites

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)

RapidNJ

Output files

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

FastTree

A newick tree is produced in

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

IQ-TREE

A newick tree is produced in

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

RAxML-NG

A newick tree is produced in

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

MultiQC

Quality statistics from the fastq files post trimmimg with fastp, bam files after mapping with bwa, and vcf files after variants are called using bcftools 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

Variant 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 pipeline_dag.dot/pipeline_dag.svg.
    • Reports generated by the pipeline: pipeline_report.html, pipeline_report.txt and software_versions.tsv.
    • 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.