Benchmarking of long read sequencing technologies and genome assembly tools

In brief

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.

Highlights

Three long read sequencing technologies were applied to the de novo assembly of a plant genome, Macadamia jansenii.
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.
A webinar presented by Valentine Murigneux and hosted by Australian Biocommons can be viewed at Webinar: Long read sequencing and assembly of Macadamia jansenii.
A webinar presented by Professor Lachlan Coin and hosted by BGI can be viewed at Webinar: Assembly of the Macadamia jansenii genome

Introduction

Sequencing technologies have advanced to the point where it is possible to generate high accuracy, haplotype resolved, chromosome scale assemblies. Several long read sequencing technologies are available on the market and a growing number of algorithms have been developed over the last years to assemble the reads generated by those technologies. When starting a new genome project, it is therefore challenging to select the most cost-effective sequencing technology as well as the most appropriate software for assembly and polishing. For this reason, it is important to benchmark different approaches applied to the same sample. 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 plant sample.

Materials and equipment

Procedure

Results

Table 1. Sequencing data

Table 2. Assembly statistics before short read polishing

Figure. BUSCO analysis of assemblies using the eudicotyledons dataset.

Figure 3. BUSCO analysis

Table 3. Gap filling for stLFR assembly

Conclusion

The three long-read technologies produced highly contiguous and complete genome assemblies.
DNA material requirements were different.
Cost associated was significantly different (May 2019) but continuous improvements in technologies have resulted in greater accuracy, increased throughput and reduced costs.
stLFR is a cheap complementary technology to ONT.
Challenges associated to the rapid evolution of technologies and softwares.
Computational resources should be considered (memory, run time): Supernova, Falcon, Pilon.

Acknowledgements

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