Workflows

Combined workflow for large genome assembly

The tutorial document for this workflow is here: https://doi.org/10.5281/zenodo.5655813

What it does: A workflow for genome assembly, containing subworkflows:

• Data QC
• Kmer counting
• Trim and filter reads
• Assembly with Flye
• Assembly polishing
• Assess genome quality

Inputs:

• long reads and short reads in fastq format
• reference genome for Quast

Outputs:

• Data information - QC, kmers
• Filtered, trimmed reads
• Genome assembly, assembly graph, ...

Assess genome quality; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Assesses the quality of the genome assembly: generate some statistics and determine if expected genes are present; align contigs to a reference genome.
• Inputs: polished assembly; reference_genome.fasta (e.g. of a closely-related species, if available).
• Outputs: Busco table of genes found; Quast HTML report, and link to Icarus contigs browser, showing contigs aligned to a reference ...

Assembly polishing subworkflow: Racon polishing with long reads

Inputs: long reads and assembly contigs

Workflow steps:

• minimap2 : long reads are mapped to assembly => overlaps.paf.
• overaps, long reads, assembly => Racon => polished assembly 1
• using polished assembly 1 as input; repeat minimap2 + racon => polished assembly 2
• using polished assembly 2 as input, repeat minimap2 + racon => polished assembly 3
• using polished assembly 3 as input, repeat minimap2 + racon => ...

Assembly with Flye; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Assembles long reads with the tool Flye
• Inputs: long reads (may be raw, or filtered, and/or corrected); fastq.gz format
• Outputs: Flye assembly fasta; Fasta stats on assembly.fasta; Assembly graph image from Bandage; Bar chart of contig sizes; Quast reports of genome assembly
• Tools used: Flye, Fasta statistics, Bandage, Bar chart, Quast
• Input parameters: None required, but recommend ...

Trim and filter reads; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Trims and filters raw sequence reads according to specified settings.
• Inputs: Long reads (format fastq); Short reads R1 and R2 (format fastq)
• Outputs: Trimmed and filtered reads: fastp_filtered_long_reads.fastq.gz (But note: no trimming or filtering is on by default), fastp_filtered_R1.fastq.gz, fastp_filtered_R2.fastq.gz
• Reports: fastp report on long reads, html; fastp report ...

Kmer counting step, can run alone or as part of a combined workflow for large genome assembly.

• What it does: Estimates genome size and heterozygosity based on counts of kmers
• Inputs: One set of short reads: e.g. R1.fq.gz
• Outputs: GenomeScope graphs
• Tools used: Meryl, GenomeScope
• Input parameters: None required
• Workflow steps: The tool meryl counts kmers in the input reads (k=21), then converts this into a histogram. GenomeScope: runs a model on the histogram; reports estimates. k-mer ...

Data QC step, can run alone or as part of a combined workflow for large genome assembly.

• What it does: Reports statistics from sequencing reads.
• Inputs: long reads (fastq.gz format), short reads (R1 and R2) (fastq.gz format).
• Outputs: For long reads: a nanoplot report (the HTML report summarizes all the information). For short reads: a MultiQC report.
• Tools used: Nanoplot, FastQC, MultiQC.
• Input parameters: None required.
• Workflow steps: Long reads are analysed by Nanoplot; Short reads ...

Assembly polishing subworkflow: Racon polishing with short reads

Inputs: short reads and assembly (usually pre-polished with other tools first, e.g. Racon + long reads; Medaka)

Workflow steps:

• minimap2: short reads (R1 only) are mapped to the assembly => overlaps.paf. Minimap2 setting is for short reads.
• overlaps + short reads + assembly => Racon => polished assembly 1
• using polished assembly 1 as input; repeat minimap2 + racon => polished assembly 2
• Racon short-read polished ...

Assembly polishing; can run alone or as part of a combined workflow for large genome assembly.

• What it does: Polishes (corrects) an assembly, using long reads (with the tools Racon and Medaka) and short reads (with the tool Racon). (Note: medaka is only for nanopore reads, not PacBio reads).
• Inputs: assembly to be polished: assembly.fasta; long reads - the same set used in the assembly (e.g. may be raw or filtered) fastq.gz format; short reads, R1 only, in fastq.gz format
• Outputs: ...

This is a Galaxy workflow that uses to convert the16S BIOM file to table and figures. It is part of the metaDEGalaxy workflow MetaDEGalaxy: Galaxy workflow for differential abundance analysis of 16s metagenomic data.