Introduction Usage Parameters 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:

Read quality check

FastQC

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

Trim reads

Trimmomatic is a software used to trim adapter sequences and low quality bases from the end of reads and quality check after this step is performed again with Fastqc.

Output files
  • fastqc/
    • *_fastqc.html: FastQC report containing quality metrics.
    • *_fastqc.zip: Zip archive containing the FastQC report, tab-delimited data file and plot images.
    • *_trim_fastqc.html: FastQC report containing quality metrics for trimmed reads.
    • *_trim_fastqc.zip: Zip archive containing the FastQC report, tab-delimited data file and plot images for trimmed reads.
Note

The FastQC plots displayed in the MultiQC report shows both untrimmed and trimmed reads so they can be directly compared.

Alignment on Reference

The alignment is performed using BWA and the aligned reads are then sorted by chromosome coordinates with samtools.

Output files
  • alignment/bwa/
    • <sample>.bam and <sample>.bam.bai

Mark Duplicate reads

Read pairs that are likely to have originated from duplicates of the same original DNA fragments through some artificial processes are identified. These are considered to be non-independent observations, so all but a single read pair within each set of duplicates are marked, not removed from the bam file.

Output files
  • alignment/markduplicates/
    • <sample>.md.bam and <sample>.md.bam.bai
  • reports/markduplicates/
    • <sample>.md.MarkDuplicates.metrics.txt

Signal track generation

deepTools is used to generate single fraction signals in bigWig format, an indexed binary format useful for displaying dense, continuous data in Genome Browsers such as the UCSC and IGV. The bigWig format is also supported by various bioinformatics software for downstream processing such as meta-profile plotting.

Output files
  • single_tracks/deeptools/
    • <sample>.bigWig

Comparisons

When --comparisonFile is set, the difference between sample1 and sample2 read density profile smoothed by the Gaussian kernel is calculated and saved in bigwig format, as described in Kharchenko PK, Tolstorukov MY, Park PJ “Design and analysis of ChIP-seq experiments for DNA-binding proteins” Nat. Biotech. doi:10.1038/nbt.1508

Output files
  • comparisons/spp_mle/
    • <sample1>.md_VS_<sample2>.md.bw

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.

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.

Reference genome files

A number of genome-specific files if required by some of the analysis steps. If the --save_reference parameter is provided then the alignment indices generated by the pipeline will be saved in this directory.

Output files
  • genome/
    • bwa/: Directory containing BWA indices.

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.