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

Abstract

Background 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, and a growing number of algorithms have been developed 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. It is thus important to benchmark different approaches applied to the same sample. Results Here, we report a comparison of 3 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 Pacific Biosciences and Nanopore reads. Results obtained from combining long-read technologies or short-read and long-read technologies are also presented. The assemblies were compared for contiguity, base accuracy, and completeness, as well as sequencing costs and DNA material requirements. Conclusions The 3 long-read technologies produced highly contiguous and complete genome assemblies of M. 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. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

Improvements in the Sequencing and Assembly of Plant Genomes

“…Recent advances in DNA sequencing technology have facilitated the sequencing and assembly of plant genomes with a rapid growth in reports of high quality chromosome level assemblies [1]. A basal eudicot, Macadamia jansenii, was used to compare the range of long read sequencing and assembly technologies available in 2019 [2]. The Pac Bio (Sequel), Oxford Nanopore Technology (PromethION) and BGI (single-tube Long Fragment Read) platforms were applied to the analysis of the same sample.…”

“…The Pac Bio (Sequel), Oxford Nanopore Technology (PromethION) and BGI (single-tube Long Fragment Read) platforms were applied to the analysis of the same sample. Assembly tools were evaluated for these data sets and the contribution of short reads to improving assemblies assessed [2].…”

“…Influence of data volume on assembly for Macadamia species N50 of contigs is plotted against the genome coverage. Genome sizes used to calculate coverage were; M.integrifolia, 895Mb[12]; M janseni, 780Mb[2]; M. tetraphylla, 758Mb[23] and M. ternifolia, 758Mb (not known but assumed the same as M. tetraphylla due to similar assembly size). Influence of data volume on assembly for diverse species.…”

Chromosome-scale assembly of theSparassis latifoliagenome obtained using long-read and Hi-C sequencing

“…For ONT data, the highest contiguity is obtained with a long-read polished assembly, as compared to a hybrid assembly incorporating both the short and long reads. ONT also significantly improves the assembly completeness as compared to the assembly generated using Illumina reads only (Murigneux et al, 2020). Besides, research on signal simulation of nanopore sequences is highly desirable for method developments of nanopore sequencing applications.…”

Optimizing experimental design for genome sequencing and assembly with Oxford Nanopore Technologies

“…Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) are the current front-runners in long-read sequencing platforms; both are capable of average read lengths in the tens of thousands of base pairs and, theoretically, entire chromosomes can be sequenced in a single read [2, 3]. Both are also capable of producing quality genome assemblies with reasonable amounts of data [4].…”