Introduction Usage Output Results

Introduction

This document describes the output produced by the nf-core/genomeqc.

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:

  • NCBI genome download - Download genomes and their annotations from RefSeq
  • Genome quality metrics:
    • Quast - Genome quality and contiguity metrics
    • tidk - Identify telomeric repeats
    • Merqury - Genome completeness and accuracy basedon raw sequecing k-mer counts
  • Annotation quality metrics:
  • GffRead - Extract longest isoform from FASTA file
  • BUSCO - Genome completeness based on single copy markers
  • Orthofinder - Phylogenetic orthology inference
  • Tree summary - Phylogenetic summary plot
  • MultiQC - Aggregate report describing results and QC from the whole pipeline
  • Pipeline information - Report metrics generated during the workflow execution

NCBI genome download

NCBI genome download is a tool for downloading assemlbies from the NCBI FTP site.

In nf-core/genomeqc, it inputs RefSeq IDs and donwloads the respective assembly and annotation in FASTA and GFF formats. If local files are provided, this step is skipped.

Output files
  • ncbigenomedownload/
    • <assembly>.fa.gz: Genome assembly in FASTA format.
    • <assembly>.gff3.gz: Annotation in GFF format.

This directory will only be present if --save_assembly flag is set.

Quast

Quast provides different quality metrics about the genome assembly. It computes contiguity stats (N50, N90), genome size, GC% content and number of sequences.

It generates a report in different formats, as well as an HTML file with in integrated contig viewer.

Output files
  • quast/<species_name>/
    • icarus.html: Contig viewr in HTML format
    • report.html: Assembly stats in HTML format
    • report.pdf: Assembly stats in tsv format
    • report.tsv: Assembly QC as HTML report

tidk

tidk is a tool to identify and visualise telomeric repeats from asseblies.

It will use a known telomeric repeat as input string, and will find occurrences of these sequence in windows across the genome.

  • tidk/
    • <species_name>.tsv: Report with the number of repeats found in different number of windows
    • <species_name>.svg: Plot with the repeat distribution

To run nf-core/genomeqc with tidk, the flag --run_tidk must be provided.

output_example_tidk

Merqury

Merqury uses k-mers from sequencing reads to evaluate the assembly quality and completness without the need of a high quality reference.

It generates a histogram relating k-mer counts in the read set to their associated counts in the assembly, as well as a completness report.

Output files
  • merqury/
    • <assembly>.html: Contig viewer in HTML format
    • <assembly>.html: Assembly stats in HTML format
    • <assembly>.pdf: Assembly stats in tsv format
    • <assembly>.tsv: Assembly QC as HTML report

To run nf-core/genomeqc with merqury, the flag --run_merqury must be provided.

AGAT sp_statistics

AGAT sp_statistics computes several annotation metrics such as number of genes, transcripts, exons, etc.

Output files
  • agat/
    • <species_name>.stats.txt: Contig viewr in HTML format

AGAT sp_keep_longest_isoform

AGAT sp_keep_longest_isoform filters GFF file to keep the longest isoform per gene. Longest isoforms are recommended as input for both BUSCO and Orthofinder.

Output files
  • longest/
    • <species_name>.longest.gff3: Contig viewr in HTML format

This directory will only be present if --save_longest_isoform flag is set.

Gene overlaps

Gene overlaps is a local module based on the R package GenomicRanges, used for manipulating genomic intervals. It finds the number of genes that are overlapping in the GFF file, which can be used as a metric to evaluate the quality of the annotation.

It outputs a brief report with information about the number of reads, the number of genes fully contained in sense direction and in the antisense direction, and the total number of overlapping genes.

Output files
  • longest/
    • Count.<species_name>.tsv: Report in tsv format

GffRead

GffRead extracts the protein sequences using the genome assembly and annoation as input.

Output files
  • gffread/
    • <species_name>.longest.fasta: Report in tsv format

This directory will only be present if --save_extracted_seqs flag is set.

BUSCO

BUSCO is a tool for assessing the quality of assemblies based on the presence of single copy orthotologues. It computes the compleness based on evolutionarily informed expectations of gene content, whether this single copy markers are present in single copy, duplicated, fragmented or absent.

It outputs a report with completness stats, a summarized table with these stats, and an ideaogram with single copy markers mapped against each chromosome or sequence.

Output files
  • busco/
    • short_summary.specific.<busco_db>.<species_name>.fasta.txt Completness report in tsv format
    • <species_name>-<busco_db>-busco.batch_summary.txt: Summarized completness report in tsv format
    • <species_name>_<lineage>.png Ideogram with the location of single copy markers

output_example_busco

Orthofinder

Orthofinder finds groups of orthologous genes and uses these orthologous genes for phylogenetic inference.

It output a rooted species tree which is later used to present the quality stats of the assemblies.

Tree summary

Tree summary is a local module that takes the rooted trees species from Orthofinder, as well as the ouput statistics from the mentioned tools.

The idea of the tree summary is to give some phylogenetic context to the quality stats, which might help users when evaluating the integrity of the assemblies.

  • tree_summary/
    • tree_plot.pdf Tree summary with quality statistics

output_example_tree

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.

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.