Biology subjects

Topp, B.

Publications and source records attributed to Topp, B..

5 recordsLinked to original sources

bioRxiv2021

De-novo chromosome level assembly of plant genomes from long read sequence data

Recent advances in the sequencing and assembly of plant genomes have allowed the generation of genomes with increasing contiguity and sequence accuracy. The chromosome level assembly of the contigs generated from long read sequencing has involved the use of proximity analysis (Hi-C) or traditional genetic maps to guide the placement of sequence contigs within chromosomes. The development of highly accurate long reads by repeated sequencing of circularized DNA (PacBio HiFi) has greatly increased the size of contigs. We now report the use of HiFiasm to assemble the genome of Macadamia jansenii. a genome that has been used as model to test sequencing and assembly. This achieved almost complete chromosome level assembly from the sequence data alone without the need for higher level chromosome map information. Eight of the 14 chromosomes were represented by a single large contig and the other 6 assembled into 2-4 main contigs. The small number of chromosome breaks appear to be due to highly repetitive regions of ribosomal genes that cannot be assembled by these approaches. De novo assembly of near complete chromosome level plant genomes now seems possible using these sequencing and assembly tools. Further targeted strategies might allow these remaining gaps to be closed. Significance statement (of up to two sentences)De novo assembly of near complete chromosome level plant genomes is now possible using current long read sequencing and assembly tools.

genomics↗

bioRxiv2021

The genome of the endangered Macadamia jansenii displays little diversity but represents an 1 important genetic resource for plant breeding

Macadamia, a recently domesticated expanding nut crop in the tropical and subtropical regions of the world, is one of the most economically important genera in the diverse and widely adapted Proteaceae family. All four species of Macadamia are rare in the wild with the most recently discovered, M. jansenii, being endangered. The M. jansenii genome has been used as a model for testing sequencing methods using a wide range of long read sequencing techniques. Here we report a chromosome level genome assembly, generated using a combination of Pacific Biosciences sequencing and Hi-C, comprising 14 pseudo-molecules, with a N50 of 58 Mb and a total 758 Mb genome assembly size of which 56% is repetitive. Completeness assessment revealed that the assembly covered 96.9% of the conserved single copy genes. Annotation predicted 31,591 protein coding genes and allowed the characterization of genes encoding biosynthesis of cyanogenic glycosides, fatty acid metabolism and anti-microbial proteins. Re-sequencing of seven other genotypes confirmed low diversity and low heterozygosity within this endangered species. Important morphological characteristics of this species such as small tree size and high kernel recovery suggest that M. jansenii is an important source of these commercial traits for breeding. As a member of a small group of families that are sister to the core eudicots, this high-quality genome also provides a key resource for evolutionary and comparative genomics studies.

genomics↗

bioRxiv2021

Improvements in the Sequencing and Assembly of Plant Genomes

BackgroundAdvances in DNA sequencing have reduced the difficulty of sequencing and assembling plant genomes. A range of methods for long read sequencing and assembly have been recently compared and we now extend the earlier study and report a comparison with more recent methods. ResultsUpdated Oxford Nanopore Technology software supported improved assemblies. The use of more accurate sequences produced by repeated sequencing of the same molecule (PacBio HiFi) resulted in much less fragmented assembly of sequencing reads. The use of more data to give increased genome coverage resulted in longer contigs (higher N50) but reduced the total length of the assemblies and improved genome completeness (BUSCO). The original model species, Macadamia jansenii, a basal eudicot, was also compared with the 3 other Macadamia species and with avocado (Persea americana), a magnoliid, and jojoba (Simmondsia chinensis) a core eudicot. In these phylogenetically diverse angiosperms, increasing sequence data volumes also caused a highly linear increase in contig size, decreased assembly length and further improved already high completeness. Differences in genome size and sequence complexity apparently influenced the success of assembly from these different species. ConclusionsAdvances in long read sequencing technology have continued to significantly improve the results of sequencing and assembly of plant genomes. However, results were consistently improved by greater genome coverage (using an increased number of reads) with the amount needed to achieve a particular level of assembly being species dependant.

genomics↗

bioRxiv2020

Chromosome-scale assembly and annotation of the macadamia genome(Macadamia integrifolia HAES 741)

Macadamia integrifolia is a representative of the large basal eudicot family Proteaceae and the main progenitor species of the Australian native nut crop macadamia. Since its commercialisation in Hawaii fewer than 100 years ago, global production has expanded rapidly. However, genomic resources are limited in comparison to other horticultural crops. The first draft assembly of M. integrifolia had good coverage of the functional gene space but its high fragmentation has restricted its use in comparative genomics and association studies. Here we have generated an improved assembly of cultivar HAES 741 (4,094 scaffolds, 745 Mb, N50 413 kb) using a combination of Illumina paired and PacBio long read sequences. Scaffolds were anchored to 14 pseudo-chromosomes using seven genetic linkage maps. This assembly has improved contiguity and coverage, with >120 Gb of additional sequence. Following annotation, 34,274 protein-coding genes were predicted, representing 92% of the expected gene content. Our results indicate that the macadamia genome is repetitive and heterozygous. The total repeat content was 55% and genome-wide heterozygosity, estimated by read mapping, was 0.98% or one SNP per 102 bp. This is the first chromosome-scale genome assembly for macadamia and the Proteaceae. It is expected to be a valuable resource for breeding, gene discovery, conservation and evolutionary genomics.

genomics↗

bioRxiv2020

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

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. Here, 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. Results obtained from combining long read technologies or short read and long read technologies are also presented. 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. We propose updating this comparison regularly with reports on significant iterations of the sequencing technologies.

genomics↗