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avantonder/bovisanalyzer: 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:

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.

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

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

Fastq summary statistics

Output files
  • fastqscan/
    • *.json: JSON formatted file of summary statistics.

fastq-scan is a tool for generating FASTQ summary statistics in JSON format.

Reference Indexing

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

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

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

Read Trimming

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

fastp is a tool used to perform adapter/quality trimming on sequencing reads.

MultiQC - fastp trimmed reads plot

Assign taxonomy to reads

Output files
  • kraken2/
    • *.kraken2.report.txt: Kraken 2 taxonomic report. See here for a detailed description of the format.

Kraken 2 is a sequence classifier that assigns taxonomic labels to DNA sequences. Kraken 2 examines the k-mers within a query sequence and uses the information within those k-mers to query a database. That database maps k-mers to the lowest common ancestor (LCA) of all genomes known to contain a given k-mer.

MultiQC - Kraken 2 classification plot

Re-estimate taxonomy

Output files
  • bracken/
    • *_S.tsv: Bracken TSV output report of the re-estimated abundances. See here for a detailed description of the format.

Bracken (Bayesian Reestimation of Abundance with KrakEN) is a highly accurate statistical method that computes the abundance of species in DNA sequences from a metagenomics sample.

Read Subsampling

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

rasusa is used to subsample reads to a depth cutoff of a default of 100.

Detect drug resistance and lineage

Output files
  • tbprofiler/results/
    • *.csv CSV formated result file of resistance and strain type
    • *.txt JSON formated result file of resistance and strain type
    • *.json Text file of resistance and strain type

TB-profiler is a profiling tool for Mycobacterium tuberculosis to detect drug resistance and lineage from WGS data

Detect spoligotype

Output files
  • spotyping/
    • *.txt Text file containing binary and octal

SpoTyping is a tool for extracting the spoligotype binary and binary from sequence reads.

Read Mapping

By default the 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

MultiQC - Kraken 2 classification plot

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

MultiQC - bcftools variants plot

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

Calculate percentage of reference mapped

The seqtk tool is used to identify the number of mapped bases within the pseudogenome fasta files.

Output files
  • seqtk/
    • *.tsv tsv with base count and distribution for each pseudogenome

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/
    • masked_alignment.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

Mask alignment

Output files
  • pseudogenomes/
    • masked_alignment.fas masked alignment of all sample pseudogenomes and the reference sequence

remove_blocks_from_aln is a tool for masking sites in an alignment using coordinates contained in the AF2122_region_exclude file in the assets directory.

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)

Summarise sample metadata

Summary files from fastqscan, bracken, spotyping, tbprofiler and seqtk are collected and a final metadata summary created

Output files
  • metadata/
    • fastq-scan_summary.tsv Summary of fastq metrics for all samples
    • mapping_summary.tsv Summary of seqtk output for all samples
    • metadata_summary.tsv Summary of important metadata for all samples
    • species_composition.tsv Taxonomic composition of reads for all samples
    • spoligotype_summary.tsv Summary of spoligotypes for all samples
    • tbprofiler.variants.txt Text file of variants for all samples
    • tbprofiler.txt Text file of lineage and variants for all samples
    • tbprofiler.lineage.itol.txt Text file in iTOL format of lineage for all samples
    • tbprofiler.json Json file of lineage and variants for all samples
    • tbprofiler.dr.itol.txt Text file in iTOL format of drug resistance profile for all samples
    • tbprofiler.dr.indiv.itol.txt Text file in iTOL format of individual drug resistance profiles for all samples

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.

MultiQC - overall 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.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.

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.