nf-core/genomeassembler

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Introduction

This document describes the output produced by 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 preparation
  • ONT Reads:
  • HiFi reads:
  • Short reads:
• Assembly, choice between assemblers
• Polishing
• Scaffolding
• Annotation liftover
• Quality control
• Reporting

Output structure

Outputs are collect into the output directory by sample:

Within each sample, the files are structured as follows:

Read preparation

The outputs from all read preparation steps are emitted into <SampleName>/reads/.

ONT reads

If the basecalls are scattered across multiple files, collect can be used to collect those into a single file. porechop is a tool that identifies and trims adapter sequences from ONT reads. genomescope estimates genome size and ploidy from the k-mer spectrum computed by jellyfish.

HiFi reads

lima performs trimming of adapters from pacbio HiFi reads.

Short reads

TrimGalore! can remove adapters from illumina short-reads. meryl calculates the k-mer spectrum of short reads.

Assembly

This folder contains the initial assemblies of the provided reads. Depending on the assembly strategy chosen, different assemblers are used. flye performs assembly of ONT reads hifiasm performs assembly of HiFi reads, or combinations of HiFi reads and ONT reads in --ul mode. ragtag performs scaffolding and can be used to scaffold assemblies of ONT onto assemblies of HiFi reads. Annotation gff3 and unmapped.txt files are only created if a reference for annotation liftover is provided and lift_annotations is enabled.

Polishing

Polishing can be used to correct errors in the assembly. This pipeline supports two polishing tools. medaka polishes assemblies using the ONT reads that were used for assembly. pilon polishes any type of assembly using short-reads. Annotation gff3 and unmapped.txt files are only created if a reference for annotation liftover is provided and lift_annotations is enabled.

Scaffolding

The (polished) assembly can be scaffolded using different tools. links performs scaffolding of the assembly using long-reads longstitch performs correction via Tigmint and scaffolding using long reads via ntLink and ARKS. Annotation gff3 and unmapped.txt files are only created if a reference for annotation liftover is provided and lift_annotations is enabled.

Quality control

All quality control files end up in QC. Below is the tree assuming that all steps of the pipeline were run:

• nanoq generates descriptive statistics of the nanopore reads. For each step three quality control tools can be run.
• QUAST provides assembly statistics (e.g. size, N50, etc. )
• BUSCO assess genome quality based on the presence of lineage-specific single-copy orthologs
• merqury compares the genome k-mer spectrum to the short-read k-mer spectrum to assess base-accuracy of the assembly.

The files and folders in the different QC folders are named based on <SampleName> and <stage>. SampleName is the sample name, and stage is one of: assembly, medaka, pilon, links, longstitch or ragtag.

Alignments

All alignments created are saved to the results directory.

Alignments are created for:

• pilon: short read alignment
• QUAST:
  • long reads against reference (if provided)
  • long reads against assemblies / polishs / scaffolds

The files in the alignment folder have the following base name structure: <SampleName>_<stage>. SampleName is the sample name, and stage is one of: assembly, medaka, pilon, links, longstitch or ragtag.

Report

The pipeline collects the quality control outputs into an html report. Below is the tree assuming that all steps of the pipeline were run:

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.