Long read sequencing

The Genome Innovation Hub continually develops a number of protocols and procedures while undertaking our collaborative projects. Below are a selection of detailed developments useful to researchers undertaking long read sequencing procedures in the laboratory.

Developments

High molecular weight nuclear DNA extraction from difficult plant species

Long read sequencing technologies require highly pure high molecular weight (HMW) intact DNA to utilise the optimal capacity of these technologies. Getting pure HMW DNA in non-model plant is a challenge due to complex cellular metabolites that chemically behave like DNA molecules. Isolation of nuclei helps to remove the majority of cellular impurities in DNA extraction processes. Recently, few protocols for HMW DNA extraction from plant species targeting to long read sequencing have been developed from different laboratories. Herein, the HMW nuclear DNA extraction method is optimised at the nuclei isolation step where the centrifugation speed is independent of genome size. Using this method, highly pure HMW nuclear DNA was extracted from two difficult plant species with a wide range of genome size – Macadamia jansenii (~800 mb) and Beauregard sweet potato (~2.4 Gb) and the quality was assessed by nanopore sequencing.

Beads-free ONT ligation kit library preparation for ultra-long read sequencing

The standard protocol of Oxford Nanopore Technologies (ONT) ligation sequencing kit (e.g. SQK-LSK109) uses AMPure beads at DNA repair and end-prep clean up and adapter clean up steps. AMPure beads either shear long fragments DNA or form clump with ultra-High Molecular Weight DNA and High Molecular Weight DNA resulting poor sequencing library preparation. In this method, AMPure beads are replaced by the PEG/NaCl solution and short read eliminator (SRE) XL kit to keep DNA intact during the sequencing library preparation. In addition, loading beads (LB) is also excluded on preparing loading library in order to prevent beads clumping. This protocol is useful to generate long 426 kb ultra-long reads with 7.2 Gb sequence throughput (in MinION) generated in 24 h.

Benchmarking of long read sequencing technologies and genome assembly tools

We report a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii. We have generated sequencing data using Pacific Biosciences (Sequel I), Oxford Nanopore Technologies (PromethION) and BGI (single-tube Long Fragment Read) technologies for the same sample. Several assemblers were benchmarked in the assembly of PacBio and Nanopore reads. The assemblies were compared for contiguity, accuracy and completeness as well as sequencing costs and DNA material requirements. Overall, the three long read technologies produced highly contiguous and complete genome assemblies of Macadamia jansenii. At the time of sequencing, the cost associated with each method was significantly different, but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs.

Featured projects	Duration
Culture-independent metagenomic diagnostics Genome Innovation Hub Collaborative Project (UQ infrastructure)	2021
Deciphering the epitranscriptome machinery using direct native RNA sequencing Genome Innovation Hub Collaborative Project (UQ infrastructure)	2021
Assessing DNA Replication Dynamics in Cancer Genome Innovation Hub Collaborative Project (UQ infrastructure)	2021
A genomic dissection of metaorganisms: molecular approaches for teasing apart the hologenome Genome Innovation Hub Collaborative Project (UQ infrastructure)	2020
Targeted capture and detection of 16s rRNA gene applying PCR free long read sequencing Genome Innovation Hub Collaborative Project (UQ infrastructure)	2020
Applying long-read sequencing technology to understand telomere repair and DNA replication Genome Innovation Hub Collaborative Project (UQ infrastructure)	2019
Assembly of complex genome (Macadamia) using single molecule sequencing Genome Innovation Hub Collaborative Project (UQ infrastructure)	2019

Publications

Comparison of long-read methods for sequencing and assembly of a plant genome

Valentine Murigneux, Subash Kumar Rai, Agnelo Furtado, Timothy J C Bruxner, Wei Tian, Ivon Harliwong, Hanmin Wei, Bicheng Yang, Qianyu Ye, Ellis Anderson, Qing Mao, Radoje Drmanac, Ou Wang, Brock A Peters, Mengyang Xu, Pei Wu, Bruce Topp, Lachlan J M Coin, Robert J Henry

GigaScience, Volume 9, Issue 12, December 2020, giaa146, https://doi.org/10.1093/gigascience/giaa146

Improvements in the Sequencing and Assembly of Plant Genomes

Priyanka Sharma, Othman Aldossary, Bader Alsubaie, Ibrahim Al-Mssallem, Onkar Nath, Neena Mitter, Gabriel Margarido, Bruce Topp, Valentine Murigneux, Ardy Masouleh, Agnelo Furtado, Robert J Henry

bioRxiv 2021.01.22.427724; doi: https://doi.org/10.1101/2021.01.22.427724

Webinars

Valentine Murigneux presents a comparison of long read methods for sequencing and assembly of a plant genome

Published on: 1 May 2020

This webinar will present a comparison of three long read sequencing technologies applied to the de novo assembly of a plant genome, Macadamia jansenii, presented by Valentine Murigneux (GIH/QFAB).
GIH Collaborative Project.

Hosted by Australian BioCommons.

Professor Lachlan Coin presents a de novo assembly of the genome of Australia’s vulnerable plant species, Macadamia jansenii

Published on: 16 April 2020

In this webinar, Professor Lachlan Coin will talk about his recent study with the Genome Innovation Hub at The University of Queensland for de novo assembly of the genome of Australia’s vulnerable plant species, Macadamia jansenii. In this study several long read sequencing technologies, including MGI’s unique single-tube Long Fragment Read (stLFR), are compared for the same sample.
GIH Collaborative Project.

Hosted by MGI

Associate Professor Scott Beatson

Associate Professor

School of Chemistry and Molecular Biosciences, Faculty of Science
Affiliate Associate Professor

UQ Centre for Clinical Research, Faculty of Medicine
Dr Tim Bruxner

Sequencing Specialist

Institute for Molecular Bioscience
Dr Cheong Xin Chan

Senior Research Fellow

School of Chemistry and Molecular Biosciences, Faculty of Science
Affiliate Research Fellow

Institute for Molecular Bioscience
Professor Paul Clarke

Director, UQ Diamantina Institute

The University of Queensland Diamantina Institute, Faculty of Medicine
Associate Professor Peter Erskine

Program Leader/Assoc Prof

Centre for Mined Land Rehabilitation, Sustainable Minerals Institute
Dr Brian Forde

Advance Queensland Industry Research Fellow

UQ Centre for Clinical Research Faculty of Medicine
Dr Agnelo Furtado

Senior Research Fellow

Centre for Nutrition and Food Sciences, Queensland Alliance for Agriculture and Food Innovation
Dr Emily Goodall

Research Officer

Institute for Molecular Bioscience
Professor Ian Henderson

Director

Institute for Molecular Bioscience
Affiliate Professor

UQ Centre for Clinical Research, Faculty of Medicine
Affiliate Professor

School of Chemistry and Molecular Biosciences, Faculty of Science
Professor Robert Henry

Institute Director

Queensland Alliance for Agriculture and Food Innovation (QAAFI)
Dr Mathew Jones

Research Fellow

The University of Queensland Diamantina Institute, Faculty of Medicine
Dr Sanjaya KC

Research Officer

Institute for Molecular Bioscience
Valentine Murigneux

Senior research assistant

Genome Innovation Hub
Bioinformatician

QCIF Facility for Advanced Bioinformatics
Dr Minh-Duy Phan

Postdoctoral Research Fellow

School of Chemistry and Molecular Biosciences, Faculty of Science
Dr Subash Rai

Research Assistant

Genome Innovation Hub
Dr Elizabeth Ross

Research Fellow

Centre for Animal Science, Queensland Alliance for Agriculture and Food Innovation
Professor Mark Schembri

Professor

School of Chemistry and Molecular Biosciences, Faculty of Science
Dr Antony Van der Ent

UQ Amplify Researcher, Senior Research Fellow

Centre for Mined Land Rehabilitation, Sustainable Minerals Institute
Professor Mark Walker

Professor

School of Chemistry and Molecular Biosciences, Faculty of Science
Isabel Warner

PhD candidate

Institute for Molecular Bioscience