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June 1, 2021

A comparison of assemblers and strategies for complex, large-genome sequencing with PacBio long reads.

PacBio sequencing holds promise for addressing large-genome complexities, such as long, highly repetitive, low-complexity regions and duplication events that are difficult to resolve with short-read technologies. Several strategies, with varying outcomes, are available for de novo sequencing and assembling of larger genomes. Using a diploid fungal genome, estimated to be ~80 Mb in size, as the basis dataset for comparison, we highlight assembly options when using only PacBio sequencing or a combined strategy leveraging data sets from multiple sequencing technologies. Data generated from SMRT Sequencing was subjected to assembly using different large-genome assemblers, and comparisons of the results will be shown. These include results generated with HGAP, Celera Assembler, MIRA, PBJelly, and other assembly tools currently in development. Improvements observed include a near 50% reduction in the number of contigs coupled with at least a doubling of contig N50 size in genome assemblies incorporating SMRT Sequencing data. We further show how incorporating long reads also highlights new challenges and missed insights of short-read assemblies arising from heterozygosity inherent in multiploid genomes.

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June 1, 2021

Resources for advanced bioinformaticians working in plant and animal genomes with SMRT Sequencing.

Significant advances in bioinformatics tool development have been made to more efficiently leverage and deliver high-quality genome assemblies with PacBio long-read data. Current data throughput of SMRT Sequencing delivers average read lengths ranging from 10-15 kb with the longest reads exceeding 40 kb. This has resulted in consistent demonstration of a minimum 10-fold improvement in genome assemblies with contig N50 in the megabase range compared to assemblies generated using only short- read technologies. This poster highlights recent advances and resources available for advanced bioinformaticians and developers interested in the current state-of-the-art large genome solutions available as open-source code from PacBio and third-party solutions, including HGAP, MHAP, and ECTools. Resources and tools available on GitHub are reviewed, as well as datasets representing major model research organisms made publically available for community evaluation or interested developers.

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June 1, 2021

High-quality, highly contiguous re-assembly of the pig genome

Many applications of high throughput sequencing rely on the availability of an accurate reference genome. Errors in the reference genome assembly increase the number of false-positives in downstream analyses. Recently, we have shown that over 33% of the current pig reference genome, Sscrofa10.2, is either misassembled or otherwise unreliable for genomic analyses. Additionally, ~10% of the bases in the assembly are Ns in gaps of an arbitrary size. Thousands of highly fragmented contigs remain unplaced and many genes are known to be missing from the assembly. Here we present a new assembly of the pig genome, Sscrofa11, assembled using 65X PacBio sequencing from T.J. Tabasco, the same Duroc sow used in the assembly of Sscrofa10.2. The PacBio reads were assembled using the Falcon assembly pipeline resulting in 3,206 contigs with an initial contig N50 of 14.5Mb. We used Sscrofa10.2 as a template to scaffold the PacBio contigs, under the assumption that its gross structure is correct, and used PBJelly to fill gaps. Additional gaps were filled using large, sequenced BACs from the original assembly. Following gap filling, the assembly has substantially improved contiguity and contains more sequence than the Sscrofa10.2 assembly. Arrow and Pilon were used to polish the assembly. The contig N50 is now 58.5Mb with 103 gaps remaining. By comparing regions of the two assemblies we show that regions with structural abnormalities we identified in Sscrofa10.2 are resolved in the new PacBio assembly.

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April 21, 2020

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions in their entirety with higher continuity and accuracy than is possible with other methods.Results We used trio binning to assemble reference genomes for two species from a single individual using an interspecies cross of yak (Bos grunniens) and cattle (Bos taurus). The high heterozygosity inherent to interspecies hybrids allowed us to confidently assign >99% of long reads from the F1 offspring to parental bins using unique k-mers from parental short reads. Both the maternal (yak) and paternal (cattle) assemblies contain over one third of the acrocentric chromosomes, including the two largest chromosomes, in single haplotigs.Conclusions These haplotigs are the first vertebrate chromosome arms to be assembled gap-free and fully phased, and the first time assemblies for two species have been created from a single individual. Both assemblies are the most continuous currently available for non-model vertebrates.MbmegabaseskbkilobasesMYAmillions of years agoMHCmajor histocompatibility complexSMRTsingle molecule real time

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April 21, 2020

Genome sequence resource for Ilyonectria mors-panacis, causing rusty root rot of Panax notoginseng.

Ilyonectria mors-panacis is a serious disease hampering the production of Panax notoginseng, an important Chinese medicinal herb, widely used for its anti-inflammatory, anti-fatigue, hepato-protective, and coronary heart disease prevention effects. Here, we report the first Illumina-Pacbio hybrid sequenced draft genome assembly of I. mors-panacis strain G3B and its annotation. The availability of this genome sequence not only represents an important tool toward understanding the genetics behind the infection mechanism of I. mors-panacis strain G3B but also will help illuminate the complexities of the taxonomy of this species.

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April 21, 2020

Genome-wide selection footprints and deleterious variations in young Asian allotetraploid rapeseed.

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.

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April 21, 2020

The genomes of pecan and Chinese hickory provide insights into Carya evolution and nut nutrition.

Pecan (Carya illinoinensis) and Chinese hickory (C. cathayensis) are important commercially cultivated nut trees in the genus Carya (Juglandaceae), with high nutritional value and substantial health benefits.We obtained >187.22 and 178.87 gigabases of sequence, and ~288× and 248× genome coverage, to a pecan cultivar (“Pawnee”) and a domesticated Chinese hickory landrace (ZAFU-1), respectively. The total assembly size is 651.31 megabases (Mb) for pecan and 706.43 Mb for Chinese hickory. Two genome duplication events before the divergence from walnut were found in these species. Gene family analysis highlighted key genes in biotic and abiotic tolerance, oil, polyphenols, essential amino acids, and B vitamins. Further analyses of reduced-coverage genome sequences of 16 Carya and 2 Juglans species provide additional phylogenetic perspective on crop wild relatives.Cooperative characterization of these valuable resources provides a window to their evolutionary development and a valuable foundation for future crop improvement. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A draft nuclear-genome assembly of the acoel flatworm Praesagittifera naikaiensis.

Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis.To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ~17-kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ~656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ~70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ~99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules.Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

A critical comparison of technologies for a plant genome sequencing project.

A high-quality genome sequence of any model organism is an essential starting point for genetic and other studies. Older clone-based methods are slow and expensive, whereas faster, cheaper short-read-only assemblies can be incomplete and highly fragmented, which minimizes their usefulness. The last few years have seen the introduction of many new technologies for genome assembly. These new technologies and associated new algorithms are typically benchmarked on microbial genomes or, if they scale appropriately, on larger (e.g., human) genomes. However, plant genomes can be much more repetitive and larger than the human genome, and plant biochemistry often makes obtaining high-quality DNA that is free from contaminants difficult. Reflecting their challenging nature, we observe that plant genome assembly statistics are typically poorer than for vertebrates.Here, we compare Illumina short read, Pacific Biosciences long read, 10x Genomics linked reads, Dovetail Hi-C, and BioNano Genomics optical maps, singly and combined, in producing high-quality long-range genome assemblies of the potato species Solanum verrucosum. We benchmark the assemblies for completeness and accuracy, as well as DNA compute requirements and sequencing costs.The field of genome sequencing and assembly is reaching maturity, and the differences we observe between assemblies are surprisingly small. We expect that our results will be helpful to other genome projects, and that these datasets will be used in benchmarking by assembly algorithm developers. © The Author(s) 2019. Published by Oxford University Press.

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April 21, 2020

LR_Gapcloser: a tiling path-based gap closer that uses long reads to complete genome assembly.

Completing a genome is an important goal of genome assembly. However, many assemblies, including reference assemblies, are unfinished and have a number of gaps. Long reads obtained from third-generation sequencing (TGS) platforms can help close these gaps and improve assembly contiguity. However, current gap-closure approaches using long reads require extensive runtime and high memory usage. Thus, a fast and memory-efficient approach using long reads is needed to obtain complete genomes.We developed LR_Gapcloser to rapidly and efficiently close the gaps in genome assembly. This tool utilizes long reads generated from TGS sequencing platforms. Tested on de novo assembled gaps, repeat-derived gaps, and real gaps, LR_Gapcloser closed a higher number of gaps faster and with a lower error rate and a much lower memory usage than two existing, state-of-the art tools. This tool utilized raw reads to fill more gaps than when using error-corrected reads. It is applicable to gaps in the assemblies by different approaches and from large and complex genomes. After performing gap-closure using this tool, the contig N50 size of the human CHM1 genome was improved from 143 kb to 19 Mb, a 132-fold increase. We also closed the gaps in the Triticum urartu genome, a large genome rich in repeats; the contig N50 size was increased by 40%. Further, we evaluated the contiguity and correctness of six hybrid assembly strategies by combining the optimal TGS-based and next-generation sequencing-based assemblers with LR_Gapcloser. A proposed and optimal hybrid strategy generated a new human CHM1 genome assembly with marked contiguity. The contig N50 value was greater than 28 Mb, which is larger than previous non-reference assemblies of the diploid human genome.LR_Gapcloser is a fast and efficient tool that can be used to close gaps and improve the contiguity of genome assemblies. A proposed hybrid assembly including this tool promises reference-grade assemblies. The software is available at http://www.fishbrowser.org/software/LR_Gapcloser/.

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April 21, 2020

The comparative genomics and complex population history of Papio baboons.

Recent studies suggest that closely related species can accumulate substantial genetic and phenotypic differences despite ongoing gene flow, thus challenging traditional ideas regarding the genetics of speciation. Baboons (genus Papio) are Old World monkeys consisting of six readily distinguishable species. Baboon species hybridize in the wild, and prior data imply a complex history of differentiation and introgression. We produced a reference genome assembly for the olive baboon (Papio anubis) and whole-genome sequence data for all six extant species. We document multiple episodes of admixture and introgression during the radiation of Papio baboons, thus demonstrating their value as a model of complex evolutionary divergence, hybridization, and reticulation. These results help inform our understanding of similar cases, including modern humans, Neanderthals, Denisovans, and other ancient hominins.

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April 21, 2020

The Genome of Armadillidium vulgare (Crustacea, Isopoda) Provides Insights into Sex Chromosome Evolution in the Context of Cytoplasmic Sex Determination.

The terrestrial isopod Armadillidium vulgare is an original model to study the evolution of sex determination and symbiosis in animals. Its sex can be determined by ZW sex chromosomes, or by feminizing Wolbachia bacterial endosymbionts. Here, we report the sequence and analysis of the ZW female genome of A. vulgare. A distinguishing feature of the 1.72 gigabase assembly is the abundance of repeats (68% of the genome). We show that the Z and W sex chromosomes are essentially undifferentiated at the molecular level and the W-specific region is extremely small (at most several hundreds of kilobases). Our results suggest that recombination suppression has not spread very far from the sex-determining locus, if at all. This is consistent with A. vulgare possessing evolutionarily young sex chromosomes. We characterized multiple Wolbachia nuclear inserts in the A. vulgare genome, none of which is associated with the W-specific region. We also identified several candidate genes that may be involved in the sex determination or sexual differentiation pathways. The A. vulgare genome serves as a resource for studying the biology and evolution of crustaceans, one of the most speciose and emblematic metazoan groups. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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