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MDL/metabolt: Output

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 control of input reads - trimming and contaminant removal
  • Assembly - Assembling trimmed reads into longer contigs
  • Mapping - Mapping preprocessed reads onto contigs
  • Binning - Clustering contigs into bins representing individual genomes
  • MultiQC - Aggregate report describing results and QC from the whole pipeline
  • Pipeline information - Report metrics generated during the workflow execution

Quality control

These steps trim away the adapter sequences present in input reads, trims away bad quality bases and, reads that are too short. FastQC is run for visualising the general quality metrics of the sequencing runs before and after trimming.

FastQC

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.

Output files
  • fastqc/
    • [sample]_[1/2]_fastqc.html: FastQC report, containing quality metrics for your untrimmed raw fastq files
    • [sample].trimmed_[1/2]_fastqc.html: FastQC report, containing quality metrics for trimmed and, if specified, filtered read files

fastp

fastp is a all-in-one fastq preprocessor for read/adapter trimming and quality control. It is used in this pipeline for trimming adapter sequences and discard low-quality reads. Its output is in the results folder and part of the MultiQC report.

Output files
  • fastp/[sample]/
    • [sample/group]_trimmed_[1/2].fastp.fastq.gz: Compressed preprocessed read file
    • [sample/group]_trimmed.fastp.html: Interactive report
    • [sample/group]_trimmed.fastp.json: Report in json format

Assembly

Trimmed (short) reads are assembled with megahit.

MEGAHIT

MEGAHIT is a single node assembler for large and complex metagenomics short reads.

Output files
  • Assembly/
    • [sample/group]_assembled.contigs.fa.gz: Compressed metagenome assembly in fasta format
    • [sample/group]_assembled.log: Log file
    • intermediate_contigs: Compressed intermediate k-mers that have been generated during the assembly process.

Mapping

Contigs are mapped onto raw reads using BWA-MEM, indexed with BWA index, and sorted with samtools sort.

BWA-MEM is a software package for mapping low-divergent sequences against a large reference genome. [SAMTOOLS-SORT]

Output files
  • mapping/
    • bwa/index -vdsvs
    • [sample/group]_mapped.bam: Binary alignment map (BAM) file containing the mapped reads
    • [sample/group]_mapped.bam.bai: BAM index file
    • [sample/group]_sorted.bam: Sorted BAM file
    • [sample/group]_mapping.log: Log file containing mapping statistics

Binning

Binning is performed using MetaBAT2, which clusters contigs into bins representing individual genomes.

MetaBAT2 is a robust metagenome binning software.

Output files
  • binning/
    • [sample/group]_bins/: Directory containing binned contigs
    • [sample/group]_binning.log: Log file containing binning statistics

MultiQC

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.

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.

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.

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.yml. The pipeline_report* files will only be present if the --email / --email_on_fail parameter's are used when running the pipeline.
    • Parameters used by the pipeline run: params.json.

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.