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

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 

Outputs

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

exeReport

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

Results

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.

preQC

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

Assembly

This folder contains final assembly results in format.

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

postqc

The postqc folder contains two sub folders

• assembly_completeness
• assembly_evaluation

assembly_completeness

This contains BUSCO evaluation results for primary and associate contig.

assembly_evaluation

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

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

Workflow summaries

Metadata

Component tools

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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

Help/FAQ/Troubleshooting

Direct training and help is available if you are new to HPC and/or new to NCI/Gadi.

• Basic information to get started with the NCI Gadi for bioinformatics can be found at https://github.com/AusARG/ABLeS/wiki/temppage.
• For NCI support, contact the NCI helpdesk directly at https://www.nci.org.au/users/nci-helpdesk
• Queue limits and structure explained at https://opus.nci.org.au/display/Help/4.+PBS+Jobs

3rd party Tutorials

A tutorial by Andrew Severin on running GenomeScope 1.0 is available here: https://github.com/AusARG/hifi-assembly-workflow.git

Improved Phased Assembler tutorial is available at https://github.com/PacificBiosciences/pbbioconda/wiki/Improved-Phased-Assembler

Busco tutorial https://wurmlab.com/genomicscourse/2016-SIB/practicals/busco/busco_tutorial

Licence(s)

MIT License

Copyright (c) 2022 AusARG

Permission is hereby granted, free of charge, to any person obtaining a copy of this software and associated documentation files (the "Software"), to deal in the Software without restriction, including without limitation the rights to use, copy, modify, merge, publish, distribute, sublicense, and/or sell copies of the Software, and to permit persons to whom the Software is furnished to do so, subject to the following conditions:

The above copyright notice and this permission notice shall be included in all copies or substantial portions of the Software.

THE SOFTWARE IS PROVIDED "AS IS", WITHOUT WARRANTY OF ANY KIND, EXPRESS OR IMPLIED, INCLUDING BUT NOT LIMITED TO THE WARRANTIES OF MERCHANTABILITY, FITNESS FOR A PARTICULAR PURPOSE AND NONINFRINGEMENT. IN NO EVENT SHALL THE AUTHORS OR COPYRIGHT HOLDERS BE LIABLE FOR ANY CLAIM, DAMAGES OR OTHER LIABILITY, WHETHER IN AN ACTION OF CONTRACT, TORT OR OTHERWISE, ARISING FROM, OUT OF OR IN CONNECTION WITH THE SOFTWARE OR THE USE OR OTHER DEALINGS IN THE SOFTWARE.

Acknowledgements/citations/credits

Jung, H. et al. Twelve quick steps for genome assembly and annotation in the classroom. PLoS Comput. Biol. 16, 1–25 (2020).

2020, G. A. W. No Title. https://ucdavis-bioinformatics-training.github.io/2020-Genome_Assembly_Workshop/kmers/kmers.

Sović, I. et al. Improved Phased Assembly using HiFi Data. (2020).

Gurevich, A., Saveliev, V., Vyahhi, N. & Tesler, G. QUAST: Quality assessment tool for genome assemblies. Bioinformatics 29, 1072–1075 (2013).

Waterhouse, R. M. et al. BUSCO applications from quality assessments to gene prediction and phylogenomics. Mol. Biol. Evol. 35, 543–548 (2018).