HiFi de novo genome assembly workflow
Version 1

Workflow Type: Nextflow

HiFi de novo genome assembly workflow

HiFi-assembly-workflow is a bioinformatics pipeline that can be used to analyse Pacbio CCS reads for de novo genome assembly using PacBio Circular Consensus Sequencing (CCS) reads. This workflow is implemented in Nextflow and has 3 major sections.

Please refer to the following documentation for detailed description of each workflow section:

  • Pre-assembly quality control (QC)
  • Assembly
  • Post-assembly QC

HiFi assembly workflow flowchart

flow chart

Quick Usage:

The pipeline has been tested on NCI Gadi and AGRF balder cluster. If needed to run on AGRF cluster, please contact us at bioinformatics@agrf.org.au. Please note for running this on NCI Gadi you need access. Please refer to Gadi guidelines for account creation and usage: these can be found at https://opus.nci.org.au/display/Help/Access.

Here is an example that can be used to run a phased assembly on Gadi:

Module load nextflow/21.04.3
nextflow run Hifi_assembly.nf –bam_folder  -profile gadi 

The workflow accepts 2 mandatory arguments:
--bam_folder     --    Full Path to the CCS bam files
-profile         --    gadi/balder/local

Please note that you can either run jobs interactively or submit jobs to the cluster. This is determined by the -profile flag. By passing the gadi tag to the profile argument, the jobs are submitted and run on the cluster.

General recommendations for using the HiFi de novo genome assembly workflow

Example local profile usage

Start a screen, submit a job, and run the workflow 
Screen -S ‘name’

qsub -I -qnormal -Pwz54 -lwalltime=48:00:00,ncpus=4,mem=200GB,storage=scratch/wz54+gdata/wz54,wd
export MODULEPATH=/apps/Modules/modulefiles:/g/data/wz54/groupResources/modules

module load nextflow/21.04.3
nextflow run /g/data/wz54/groupResources/scripts/pl/hifi_assembly.nf  --bam_folder   -profile local

#This load the scripts directory to the environmental PATH and load nextflow module
module load hifi_assembly/1.0.0 


Pipeline generates various files and folders here is a brief description: The pipeline creates a folder called secondary_analysis that contains two sub folders named:

  • exeReport
  • Results -- Contains preQC, assembly and postQC analysis files


This folder contains a computation resource usage summary in various charts and a text file. report.html provides a comprehensive summary.


The Results folder contains three sub-directories preQC, assembly and postqc. As the name suggests, outputs from the respective workflow sections are placed in each of these folders.


The following table contains list of files and folder from preQC results

Output folder/file File Description
.fa Bam files converted to fasta format
kmer_analysis Folder containing kmer analysis outputs
.jf k-mer counts from each sample
.histo histogram of k-mer occurrence
genome_profiling genomescope profiling outputs
summary.txt Summary metrics of genome scope outputs
linear_plot.png Plot showing no. of times a k-mer observed by no. of k-mers with that coverage


This folder contains final assembly results in format.

  • _primary.fa - Fasta file containing primary contigs
  • _associate.fa - Fasta file containing associated contigs


The postqc folder contains two sub folders

  • assembly_completeness
  • assembly_evaluation


This contains BUSCO evaluation results for primary and associate contig.


Assembly evaluation folder contains various file formats, here is a brief description for each of the outputs.

File Description
report.txt Assessment summary in plain text format
report.tsv Tab-separated version of the summary, suitable for spreadsheets (Google Docs, Excel, etc)
report.tex LaTeX version of the summary
icarus.html Icarus main menu with links to interactive viewers
report.html HTML version of the report with interactive plots inside

Infrastructure usage and recommendations

NCI facility access

One should have a user account set with NCI to access gadi high performance computational facility. Setting up a NCI account is mentioned in detail at the following URL: https://opus.nci.org.au/display/Help/Setting+up+your+NCI+Account

Documentation for a specific infrastructure should go into a infrastructure documentation template https://github.com/AustralianBioCommons/doc_guidelines/blob/master/infrastructure_optimisation.md

Compute resource usage across tested infrastructures

Computational resource for plant case study
Process duration
Converting bam to fasta for sample 12m 54s
Generating k-mer counts and histogram 26m 43s
Profiling genome characteristics 34.7s
Denovo assembly 6h 51m 15s
evaluate_assemblies 5m 18s
assemblies_completeness 25m 57s
Computational resource for bird case study
Process duration
Converting bam to fasta for sample 12m 54s
Generating k-mer counts and histogram 26m 43s
Profiling genome characteristics 34.7s
De novo assembly 6h 51m 15s
evaluate assemblies 5m 18s
assemblies completeness 25m 57s

Workflow summaries


Metadata field Pre-assembly quality control Primary assembly Post-assembly quality control
Version 1.0 1.0 1.0
Maturity Production Production production
Creators Naga, Kenneth Naga, Kenneth Naga, Kenneth
Source AusARG/hifi-assembly-workflow
Workflow manager NextFlow NextFlow NextFlow
Container No containers used No containers used No containers used
Install method Manual Manual Manual

Component tools

Workflow element Workflow element version Workflow title
Samtools, jellyfish, genomescope 1.0 Pre-assembly quality control
Improved phased assembler (pbipa) 1.0 Primary assembly
Quast and busco 1.0 Post-assembly quality control

Required (minimum) inputs/parameters

PATH to HIFI bam folder is the minimum requirement for the processing the pipeline.

Third party tools / dependencies

The following packages are used by the pipeline.

  • nextflow/21.04.3
  • samtools/1.12
  • jellyfish/2.3.0
  • genomescope/2.0
  • ipa/1.3.1
  • quast/5.0.2
  • busco/5.2.2

The following paths contain all modules required for the pipeline.

  • /apps/Modules/modulefiles
  • /g/data/wz54/groupResources/modules

General recommendations

A more detailed Module and workflow descriptions are made avaiable

Resources available here

This repository contains structured documentation for [dependencies and usage], including links to existing repositories and community resources, as well as a description of the optimisations achieved on the following compute systems:


This work is supported by the Australian BioCommons via Bioplatforms Australia funding, the Australian Research Data Commons (https://doi.org/10.47486/PL105) and the Queensland Government RICF programme. Bioplatforms Australia and the Australian Research Data Commons are enabled by the National Collaborative Research Infrastructure Strategy (NCRIS).

The documentation in this repository is based on Australian BioCommons guidelines.

Version History

master @ d3d3d39 (earliest) Created 10th May 2022 at 00:42 by Naga Kasinadhuni

Merge pull request #2 from AusARG/jb018-recommendations

added background recommendations document

Frozen master d3d3d39
help Creators and Submitter
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Views: 134

Created: 10th May 2022 at 00:42

Last updated: 25th May 2022 at 04:15

Last used: 28th May 2022 at 04:30

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