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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 an all-in-one fastq preprocessor for read/adapter trimming and quality control. It is used in this pipeline for trimming adapter sequences and discarding 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 fast and memory-efficient assembler designed for assembling large and complex metagenomics datasets. It uses succinct de Bruijn graphs to assemble contigs from 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 generated during the assembly process.

Mapping

BWA Index

BWA Index indexes the reference genome to prepare it for alignment. This step is essential for efficient read mapping.

Output files
  • bwa_index/[sample]/
    • [sample]_indexed: Indexed reference genome files

BWA MEM

BWA MEM is a widely used tool for aligning sequencing reads to a reference genome. It is optimized for high-quality reads and supports paired-end alignment.

Output files
  • bwa_align_sorted/[sample]/
    • [sample].bam: Binary alignment map (BAM) file containing the mapped reads

SAMTOOLS Index

SAMTOOLS Index creates an index for BAM files, enabling efficient random access to alignments.

Output files
  • samtools/indexed/[sample]/
    • [sample].bai: BAM index file

Binning

MetaBAT2 JGI Summarize Bam Contig Depths

MetaBAT2 JGI Summarize Bam Contig Depths calculates the depth of coverage for each contig in a BAM file. This information is used for binning contigs into genome bins.

Output files
  • metabat2/depths/
    • [sample]_depth.txt.gz: Compressed depth information

MetaBAT2

MetaBAT2 is a tool for binning contigs into genome bins based on sequence composition and coverage depth. It is widely used for metagenomic binning.

Output files
  • metabat2/bins/[sample]/
    • tooShort/*.tooShort.fa.gz: Contigs too short for binning
    • lowDepth/*.lowDepth.fa.gz: Contigs with low depth
    • unbinned/*.unbinned.fa.gz: Unbinned contigs
    • membership/*.tsv.gz: Membership information
    • bins/*.fa.gz: Binned contigs

MultiQC

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.

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

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.