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Post-genome assembly quality control workflow using Quast, BUSCO, Meryl, Merqury and Fasta Statistics

PacBio HiFi genome assembly using hifiasm v2.1

General usage recommendations

Please see the Genome assembly with hifiasm on Galaxy Australia guide.

See change log

Acknowledgements

The workflow & the doc_guidelines template used are supported by the Australian BioCommons via Bioplatforms Australia funding, the Australian ...

Purge-duplicates-from-hifiasm-assembly

General recommendations for using Purge-duplicates-from-hifiasm-assembly

Please see the Genome assembly with hifiasm on Galaxy Australia guide.

Acknowledgements

The workflow & the doc_guidelines template used are supported by the Australian BioCommons via Bioplatforms Australia funding, the Australian ...

BAM-to-FASTQ-QC

General recommendations for using BAM-to-FASTQ-QC

Please see the Genome assembly with hifiasm on Galaxy Australia guide.

Acknowledgements

The workflow & the doc_guidelines template used are supported by the Australian BioCommons via Bioplatforms Australia funding, the Australian Research Data Commons (https://doi.org/10.47486/PL105) ...

workflow-partial-gstacks-populations

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

This workflow is part of the reference-guided stacks workflow, https://workflowhub.eu/workflows/347

This workflow takes in bam files and a population map.

To generate bam files see: https://workflowhub.eu/workflows/351

workflow-partial-bwa-mem

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

This workflow is part of the reference-guided stacks workflow, https://workflowhub.eu/workflows/347

Inputs

• demultiplexed reads in fastq format, may be output from the QC workflow. Files are in a collection.
• reference genome in fasta format ...

workflow-partial-cstacks-sstacks-gstacks

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

This workflow takes in ustacks output, and runs cstacks, sstacks and gstacks.

To generate ustacks output see https://workflowhub.eu/workflows/349

For the full de novo workflow see https://workflowhub.eu/workflows/348

workflow-partial-ustacks-only

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

For the full de novo workflow see https://workflowhub.eu/workflows/348

You may want to run ustacks with different batches of samples.

• To be able to combine these later, there are some necessary steps - we need to keep track of how many ...

workflow-denovo-stacks

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

Inputs

• demultiplexed reads in fastq format, may be output from the QC workflow. Files are in a collection.
• population map in text format

Steps and outputs

ustacks:

• input reads go to ustacks.
• ustacks assembles the reads into matching ...

workflow-ref-guided-stacks

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

Inputs

• demultiplexed reads in fastq format, may be output from the QC workflow. Files are in a collection.
• population map in text format
• reference genome in fasta format

Steps and outputs

BWA MEM 2:

• The reads are mapped to the ...

workflow-qc-of-radseq-reads

These workflows are part of a set designed to work for RAD-seq data on the Galaxy platform, using the tools from the Stacks program.

Galaxy Australia: https://usegalaxy.org.au/

Stacks: http://catchenlab.life.illinois.edu/stacks/

Inputs

• demultiplexed reads in fastq format, in a collection
• two adapter sequences in fasta format, for input into cutadapt

Steps and outputs

The workflow can be modified to suit your own parameters.

The workflow steps are:

• Run ...

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: ...