Introduction Usage Parameters Output Results Releases

Introduction

This document describes the output produced by the pipeline. Most of the plots are taken from the MultiQC report, which summarises results at the end of 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:

Quality trimming and QC

Short Read Trimming

This step quality trims the end of reads, removes degenerate or too short reads and if needed, combines reads coming from multiple sequencing runs.

Output files
  • trimming/shortreads/
    • *.fastp.fastq.gz: The trimmed/modified/unmerged fastq reads

Short Read RAW QC

FastQC gives general quality metrics about your sequenced reads. It provides information about the quality score distribution across your reads, per base sequence content (%A/T/G/C), adapter contamination and overrepresented sequences. For further reading and documentation see the FastQC help pages.

MultiQC - FastQC sequence counts plot

MultiQC - FastQC mean quality scores plot

MultiQC - FastQC adapter content plot

Note

The FastQC plots displayed in the MultiQC report shows untrimmed reads. They may contain adapter sequence and potentially regions with low quality.

Output files
  • FastQC/
    • *.html: FastQC report containing quality metrics.
    • *.zip: Zip archive containing the FastQC report, tab-delimited data file and plot images.

FastQC report

Long Read Trimming

This step performs long read trimming on Nanopore input (if provided).

Output files
  • trimming/longreads/
    • *.fastq.gz: The trimmed FASTQ file
    • *.log*: Log file

Long Read RAW QC

These steps perform long read QC for input data (if provided).

Please refer to the documentation of NanoPlot and PycoQC if you want to know more about the plots created by these tools.

Output files

  • QC_Longreads/NanoPlot: Various plots in HTML and PNG format

  • QC_Longreads/PycoQC

    • *_pycoqc.html: QC report in HTML format
    • *_pycoqc.json: QC report in JSON format

Example plot from Nanoplot:

Nanoplot

Taxonomic classification

This QC step classifies your reads using Kraken2 a k-mer based approach. This helps to identify samples that have purity issues. Ideally you will not want to assemble reads from samples that are contaminated or contain multiple species. If you like to visualize the report, try Pavian or Krakey.

Output files
  • Kraken2/
    • *.kraken2.report.txt: Classification of short reads in the Kraken(1) report format.
    • *_longreads.kraken2.report.txt: Classification of long reads in the Kraken(1) report format.

See webpage for more details.

Exemplary Kraken2 report screenshot:

Kraken2 report

Assembly Output

Trimmed reads are assembled with Unicycler in short or hybrid assembly modes. For long-read assembly, there are also canu and miniasm available. Unicycler is a pipeline on its own, which at least for Illumina reads mainly acts as a frontend to Spades with added polishing steps.

Output files
  • Unicycler/
    • *.scaffolds.fa: Final assembly in fasta format
    • *.assembly.gfa: Final assembly in Graphical Fragment Assembly (GFA) format
    • *.unicycler.log: Log file summarizing steps and intermediate results on the Unicycler execution

Check out the Unicycler documentation for more information on Unicycler output.

  • Canu/
    • *.contigs.fasta.gz: Final assembly in fasta format
    • *.report: Log file summarizing steps and intermediate results

Check out the Canu documentation for more information on Canu output.

  • Miniasm/
    • *.fasta.gz: Assembly in Fasta format
    • *_assembly_consensus.fasta.gz: Consensus assembly in fasta format (polished by Racon)

Check out the Miniasm documentation for more information on Miniasm output.

Polished assemblies

Long reads assemblies can be polished using Medaka or NanoPolish with Fast5 files.

Output files

  • Medaka/*_polished_genome.fa

    • *_polished_genome.fa: Polished consensus assembly in fasta format
    • calls_to_draft.bam: Alignment in bam format
    • calls_to_draft.bam.bai: Index of alignment
    • consensus.fasta.gaps_in_draft_coords.bed
    • consensus_probs.hdf

  • Nanopolish/

    • polished_genome.fa: Polished consensus assembly in fasta format

Assembly QC with QUAST

The assembly QC is performed with QUAST for all assemblies in one report. It reports multiple metrics including number of contigs, N50, lengths etc in form of an html report. It further creates an HTML file with integrated contig viewer (Icarus).

Output files
  • QUAST
    • report.tsv: QUAST’s report in text format
  • QUAST/report
    • icarus.html: QUAST’s contig browser as HTML
    • report.html: QUAST assembly QC as HTML report
    • report.pdf: QUAST assembly QC as pdf

QUAST QC

Icarus

Annotation

By default, the assembly is annotated with Prokka which acts as frontend for several annotation tools and includes rRNA and ORF predictions. Alternatively, on request, the assembly is annotated with Bakta or DFAST.

Output files
  • Prokka/{ID}/
    • *.gff: Annotation in gff format
    • *.txt: Annotation in text format
    • *.faa: Protein sequences in fasta format

See Prokka’s documentation for a full description of all output files.

Prokka annotation

  • Bakta/{ID}/
    • *.gff3: Annotations in gff3 format
    • *.txt: Summary in txt format
    • *.faa: CDS/sORF amino acid sequences in fasta format

See Baktas’s documentation for a full description of all output files.

  • DFAST/{ID}_results/
    • genome.gff: Annotation in gff format
    • statistics.txt: Annotation statistics in text format
    • protein.faa: Protein sequences in fasta format

Report

Some pipeline results are visualised by MultiQC, which is a visualisation tool that generates a single HTML report summarising all samples in your project. Further statistics are available in within the report data directory.

MultiQC is a visualization tool that generates a single HTML report summarising all samples in your project. Most of the pipeline QC results are visualised in the report and further statistics are available in the report data directory.

The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability.

Results generated by MultiQC collate pipeline QC from supported tools e.g. FastQC. The pipeline has special steps which also allow the software versions to be reported in the MultiQC output for future traceability. For more information about how to use MultiQC reports, see http://multiqc.info.

Output files
  • multiqc/
    • multiqc_report.html: a standalone HTML file that can be viewed in your web browser.
    • multiqc_data/: directory containing parsed statistics from the different tools used in the pipeline.
    • multiqc_plots/: directory containing static images from the report in various formats.

Pipeline information

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.

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.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter’s are used when running the pipeline.
    • Reformatted samplesheet files used as input to the pipeline: samplesheet.valid.csv.
    • Parameters used by the pipeline run: params.json.
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