nf-core/mag

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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
• Taxonomic classification of trimmed reads
• Assembly of trimmed reads
• Binning of assembled contigs
• Taxonomic classification of binned genomes
• Additional summary for binned genomes
• MultiQC - aggregate report, describing results of the whole pipeline
• Pipeline information - Report metrics generated during the workflow execution

Note that when specifying the parameter --coassemble_group, for the corresponding output filenames/directories of the assembly or downsteam processes the group ID, or more precisely the term group-[group_id], will be used instead of the sample ID.

Quality control

These steps trim away the adapter sequences present in input reads, trims away bad quality bases and sicard reads that are too short. It also removes host contaminants and sequencing controls, such as PhiX or the Lambda phage. 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.

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.

Remove PhiX sequences from short reads

The pipeline uses bowtie2 to map the reads against PhiX and removes mapped reads.

Host read removal

The pipeline uses bowtie2 to map short reads against the host reference genome specified with --host_genome or --host_fasta and removes mapped reads. The information about discarded and retained reads is also included in the MultiQC report.

Remove Phage Lambda sequences from long reads

The pipeline uses Nanolyse to map the reads against the Lambda phage and removes mapped reads.

Filtlong and porechop

The pipeline uses filtlong and porechop to perform quality control of the long reads that are eventually provided with the TSV input file.

No direct host read removal is performed for long reads. However, since within this pipeline filtlong uses a read quality based on k-mer matches to the already filtered short reads, reads not overlapping those short reads might be discarded. The lower the parameter --longreads_length_weight, the higher the impact of the read qualities for filtering. For further documentation see the filtlong online documentation.

Quality visualisation for long reads

NanoPlot is used to calculate various metrics and plots about the quality and length distribution of long reads. For more information about NanoPlot see the online documentation.

Taxonomic classification of trimmed reads

Kraken

Kraken2 classifies reads using a k-mer based approach as well as assigns taxonomy using a Lowest Common Ancestor (LCA) algorithm.

Centrifuge

Centrifuge is commonly used for the classification of DNA sequences from microbial samples. It uses an indexing scheme based on the Burrows-Wheeler transform (BWT) and the Ferragina-Manzini (FM) index.

More information on the Centrifuge website

Assembly

Trimmed (short) reads are assembled with both megahit and SPAdes. Hybrid assembly is only supported by SPAdes.

MEGAHIT

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

SPAdes

SPAdes was originally a single genome assembler that later added support for assembling metagenomes.

SPAdesHybrid

SPAdesHybrid is a part of the SPAdes software and is used when the user provides both long and short reads.

Metagenome QC with QUAST

QUAST is a tool that evaluates metagenome assemblies by computing various metrics. The QUAST output is also included in the MultiQC report, as well as in the assembly directories themselves.

Binning

Contig sequencing depth

Sequencing depth per contig and sample is generated by jgi_summarize_bam_contig_depths --outputDepth. The values correspond to (sum of exactely aligned bases) / ((contig length)-2*75). For example, for two reads aligned exactly with 10 and 9 bases on a 1000 bp long contig the depth is calculated by (10+9)/(1000-2*75) (1000bp length of contig minus 75bp from each end, which is excluded).

MetaBAT2

MetaBAT2 recovers genome bins (that is, contigs/scaffolds that all belongs to a same organism) from metagenome assemblies.

All the files and contigs in this folder will be assessed by QUAST and BUSCO.

All the files in this folder contain small and/or unbinned contigs that are not further processed.

Files in these two folders contain all contigs of an assembly.

Bin sequencing depth

For each genome bin the median sequencing depth is computed based on the corresponding contig depths given in GenomeBinning/[assembler]-[sample/group]-depth.txt.gz.

QC for metagenome assembled genomes with QUAST

QUAST is a tool that evaluates genome assemblies by computing various metrics. The QUAST output is also included in the MultiQC report, as well as in the assembly directories themselves.

QC for metagenome assembled genomes with BUSCO

BUSCO is a tool used to assess the completeness of a genome assembly. It is run on all the genome bins and high quality contigs obtained by MetaBAT2. By default, BUSCO is run in automated lineage selection mode in which it first tries to select the domain and then a more specific lineage based on phylogenetic placement. If available, result files for both the selected domain lineage and the selected more specific lineage are placed in the output directory. If a lineage dataset is specified already with --busco_reference, only results for this specific lineage will be generated.

If the parameter --save_busco_reference is set, additionally the used BUSCO lineage datasets are stored in the output directy.

Besides the reference files or output files created by BUSCO, the following summary files will be generated:

Taxonomic classification of binned genomes

CAT

CAT is a toolkit for annotating contigs and bins from metagenome-assembled-genomes. The MAG pipeline uses CAT to assign taxonomy to genome bins based on the taxnomy of the contigs.

If the parameters --cat_db_generate and --save_cat_db are set, additionally the generated CAT database is stored:

GTDB-Tk

GTDB-Tk is a toolkit for assigning taxonomic classifications to bacterial and archaeal genomes based on the Genome Database Taxonomy GTDB. nf-core/mag uses GTDB-Tk to classify binned genomes which satisfy certain quality criteria (i.e. completeness and contamination assessed with the BUSCO analysis).

Additional summary for binned genomes

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.

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.