Single Molecule, Real-Time (SMRT) Sequencing provides efficient, streamlined solutions to address new frontiers in plant genomes and transcriptomes. Inherent challenges presented by highly repetitive, low-complexity regions and duplication events are directly addressed with multi- kilobase read lengths exceeding 8.5 kb on average, with many exceeding 20 kb. Differentiating between transcript isoforms that are difficult to resolve with short-read technologies is also now possible. We present solutions available for both reference genome and transcriptome research that best leverage long reads in several plant projects including algae, Arabidopsis, rice, and spinach using only the PacBio platform. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. We will share highlights from our genome projects using the latest P5- C3 chemistry to generate high-quality reference genomes with the highest contiguity, contig N50 exceeding 1 Mb, and average base quality of QV50. Additionally, the value of long, intact reads to provide a no-assembly approach to investigate transcript isoforms using our Iso-Seq protocol will be presented for full transcriptome characterization and targeted surveys of genes with complex structures. PacBio provides the most comprehensive assembly with annotation when combining offerings for both genome and transcriptome research efforts. For more focused investigation, PacBio also offers researchers opportunities to easily investigate and survey genes with complex structures.
Since the advent of Next-Generation Sequencing (NGS), the cost of de novo genome sequencing and assembly have dropped precipitately, which has spurred interest in genome sequencing overall. Unfortunately the contiguity of the NGS assembled sequences, as well as the accuracy of these assemblies have suffered. Additionally, most NGS de novo assemblies leave large portions of genomes unresolved, and repetitive regions are often collapsed. When compared to the reference quality genome sequences produced before the NGS era, the new sequences are highly fragmented and often prove to be difficult to properly annotate. In some cases the contiguous portions are smaller than the average gene size making the sequence not nearly as useful for biologists as the earlier reference quality genomes including of Human, Mouse, C. elegans, or Drosophila. Recently, new 3rd generation sequencing technologies, long-range molecular techniques, and new informatics tools have facilitated a return to high quality assembly. We will discuss the capabilities of the technologies and assess their impact on assembly projects across the tree of life from small microbial and fungal genomes through large plant and animal genomes. Beyond improvements to contiguity, we will focus on the additional biological insights that can be made with better assemblies, including more complete analysis genes in their flanking regulatory context, in-depth studies of transposable elements and other complex gene families, and long-range synteny analysis of entire chromosomes. We will also discuss the need for new algorithms for representing and analyzing collections of many complete genomes at once.
Rebecca Johnson, director of the Australian Museum Research Institute presents finding from de novo sequencing of the koala genome. Using PacBio sequencing the Koala Genome Consortium obtained an assembly with…
PAG Conference: Long-read sequencing reveals complex genomic architecture in independent carnivorous plant lineages
In this PAG 2018 presentation, Tanya Renner of Pennsylvania State University shares research using PacBio SMRT Sequencing to understand the genomes and transcriptomes of carnivorous plants. She describes the humped…
User Group Meeting: New genotype to phenotype associations in viral metagenomes enabled by SMRT Sequencing
In this PacBio User Group Meeting lightning talk, Shawn Polson of the University of Delaware speaks about viral metagenomes, which are more challenging to distinguish than their bacterial counterparts because…
Understanding interactions among plants and the complex communities of organisms living on, in and around them requires more than one experimental approach. A new method for de novo metagenome assembly,…
During a PAG 2020 workshop, Zev Kronenberg, a senior bioinformatics engineer at PacBio, describes how he used the PacBio Iso-Seq transcriptome sequencing and analysis method to annotate great ape genomes,…
Forest tree species are increasingly subject to severe mortalities from exotic pests, diseases, and invasive organisms, accelerated by climate change. Forest health issues are threatening multiple species and ecosystem sustainability globally. While sources of resistance may be available in related species, or among surviving trees, introgression of resistance genes into threatened tree species in reasonable time frames requires genome-wide breeding tools. Asian species of chestnut (Castanea spp.) are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome with chromosome-scale sequences for Chinese chestnut (C. mollissima), the disease-resistance donor for American chestnut restoration. We also demonstrate the value of the genome as a platform for research and species restoration, including new insights into the evolution of blight resistance in Asian chestnut species, the locations in the genome of ecologically important signatures of selection differentiating American chestnut from Chinese chestnut, the identification of candidate genes for disease resistance, and preliminary comparisons of genome organization with related species.
In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a brief introduction to the basic concepts of genome assembly, annotation and metrics for evaluating the quality of draft assemblies. We then provide an overview of genome information for numerous insect species, highlighting examples from prominent model organisms, agricultural pests and disease vectors. We also introduce the major insect genome databases. The increasing availability of insect genomic resources is beneficial for developing alternative pest control methods. However, many opportunities remain for developing data-mining tools that make maximal use of the available insect genome resources. Although rapid progress has been achieved, many challenges remain in the field of insect genomics. © 2019 The Royal Entomological Society.
Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy was affected by heterozygosity of the individual sequenced, with higher accuracy for cattle and zebra finch (>97%) compared to human (82%). In addition, scaffolding with the same Hi-C chromatin contact data resulted in phased chromosome-scale scaffolds.
Centromere-mediated chromosome break drives karyotype evolution in closely related Malassezia species
Intra-chromosomal or inter-chromosomal genomic rearrangements often lead to speciation. Loss or gain of a centromere leads to alterations in chromosome number in closely related species. Thus, centromeres can enable tracing the path of evolution from the ancestral to a derived state. The Malassezia species complex of the phylum Basiodiomycota shows remarkable diversity in chromosome number ranging between six and nine chromosomes. To understand these transitions, we experimentally identified all eight centromeres as binding sites of an evolutionarily conserved outer kinetochore protein Mis12/Mtw1 in M. sympodialis. The 3 to 5 kb centromere regions share an AT-rich, poorly transcribed core region enriched with a 12 bp consensus motif. We also mapped nine such AT-rich centromeres in M. globosa and the related species Malassezia restricta and Malassezia slooffiae. While eight predicted centromeres were found within conserved synteny blocks between these species and M. sympodialis, the remaining centromere in M. globosa (MgCEN2) or its orthologous centromere in M. slooffiae (MslCEN4) and M. restricta (MreCEN8) mapped to a synteny breakpoint compared with M. sympodialis. Taken together, we provide evidence that breakage and loss of a centromere (CEN2) in an ancestral Malassezia species possessing nine chromosomes resulted in fewer chromosomes in M. sympodialis. Strikingly, the predicted centromeres of all closely related Malassezia species map to an AT-rich core on each chromosome that also shows enrichment of the 12 bp sequence motif. We propose that centromeres are fragile AT-rich sites driving karyotype diversity through breakage and inactivation in these and other species.
A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to exhibit two LGTs, and screen it for other grass-to-grass LGTs using genomic data from 146 other grass species. Through stringent phylogenomic analyses, we discovered 57 additional LGTs in the A. semialata nuclear genome, involving at least nine different donor species. The LGTs are clustered in 23 laterally acquired genomic fragments that are up to 170 kb long and have accumulated during the diversification of Alloteropsis. The majority of the 59 LGTs in A. semialata are expressed, and we show that they have added functions to the recipient genome. Functional LGTs were further detected in the genomes of five other grass species, demonstrating that this process is likely widespread in this globally important group of plants. LGT therefore appears to represent a potent evolutionary force capable of spreading functional genes among distantly related grass species. Copyright © 2019 the Author(s). Published by PNAS.
Strengths and potential pitfalls of hay-transfer for ecological restoration revealed by RAD-seq analysis in floodplain Arabis species
Achieving high intraspecific genetic diversity is a critical goal in ecological restoration as it increases the adaptive potential and long-term resilience of populations. Thus, we investigated genetic diversity within and between pristine sites in a fossil floodplain and compared it to sites restored by hay-transfer between 1997 and 2014. RAD-seq genotyping revealed that the stenoecious flood-plain species Arabis nemorensis is co-occurring with individuals that, based on ploidy, ITS-sequencing and morphology, probably belong to the close relative Arabis sagittata, which has a documented preference for dry calcareous grasslands but has not been reported in floodplain meadows. We show that hay-transfer maintains genetic diversity for both species. Additionally, in A. sagittata, transfer from multiple genetically isolated pristine sites resulted in restored sites with increased diversity and admixed local genotypes. In A. nemorensis, transfer did not create novel admixture dynamics because genetic diversity between pristine sites was less differentiated. Thus, the effects of hay-transfer on genetic diversity also depend on the genetic makeup of the donor communities of each species, especially when local material is mixed. Our results demonstrate the efficiency of hay-transfer for habitat restoration and emphasize the importance of pre-restoration characterization of micro-geographic patterns of intraspecific diversity of the community to guarantee that restoration practices reach their goal, i.e. maximize the adaptive potential of the entire restored plant community. Overlooking these patterns may alter the balance between species in the community. Additionally, our comparison of summary statistics obtained from de novo and reference-based RAD-seq pipelines shows that the genomic impact of restoration can be reliably monitored in species lacking prior genomic knowledge.
Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from textquoteleftfinishedtextquoteright. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm and complement the synteny-based adjacencies: six with physical mapping data that anchor scaffolds to chromosome locations, 13 with paired-end RNA sequencing (RNAseq) data, and three with new assemblies based on re-scaffolding or Pacific Biosciences long-read data. Our combined analyses produced 20 new superscaffolded assemblies with improved contiguities: seven for which assignments of non-anchored scaffolds to chromosome arms span more than 75% of the assemblies, and a further seven with chromosome anchoring including an 88% anchored Anopheles arabiensis assembly and, respectively, 73% and 84% anchored assemblies with comprehensively updated cytogenetic photomaps for Anopheles funestus and Anopheles stephensi.Conclusions Experimental data from probe mapping, RNAseq, or long-read technologies, where available, all contribute to successful upgrading of draft assemblies. Our comparisons show that gene synteny-based computational methods represent a valuable alternative or complementary approach. Our improved Anopheles reference assemblies highlight the utility of applying comparative genomics approaches to improve community genomic resources.ADADSEQAGOAGOUTI-basedAGOUTIannotated genome optimization using transcriptome information toolALNalignment-basedCAMSAcomparative analysis and merging of scaffold assemblies toolDPdynamic programmingFISHfluorescence in situ hybridizationGAGOS-ASMGOS-ASMGene order scaffold assemblerKbpkilobasepairsMbpmegabasepairsOSORTHOSTITCHPacBioPacific BiosciencesPBPacBio-basedPHYphysical-mapping-basedRNAseqRNA sequencingQTLquantitative trait lociSYNsynteny-based.
Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently significantly diverged, with rapid genome differentiation after hybridization and intensive local selective breeding. The first historical introgression of B. rapa dramatically broadened the allelic pool but decreased the deleterious variations of Asian rapeseed. The second historical introgression of the double-low traits of European rapeseed (canola) has reshaped Asian rapeseed into two groups (double-low and double-high), accompanied by an increase in genetic load in the double-low group. This study demonstrates distinctive genomic footprints and deleterious SNP (single nucleotide polymorphism) variants for local adaptation by recent intra- and interspecies introgression events and provides novel insights for understanding the rapid genome evolution of a young allopolyploid crop. © 2019 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.