Menu
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.


April 21, 2020

Genome assembly and annotation of the Trichoplusia ni Tni-FNL insect cell line enabled by long-read technologies.

Trichoplusiani derived cell lines are commonly used to enable recombinant protein expression via baculovirus infection to generate materials approved for clinical use and in clinical trials. In order to develop systems biology and genome engineering tools to improve protein expression in this host, we performed de novo genome assembly of the Trichoplusiani-derived cell line Tni-FNL.By integration of PacBio single-molecule sequencing, Bionano optical mapping, and 10X Genomics linked-reads data, we have produced a draft genome assembly of Tni-FNL.Our assembly contains 280 scaffolds, with a N50 scaffold size of 2.3 Mb and a total length of 359 Mb. Annotation of the Tni-FNL genome resulted in 14,101 predicted genes and 93.2% of the predicted proteome contained recognizable protein domains. Ortholog searches within the superorder Holometabola provided further evidence of high accuracy and completeness of the Tni-FNL genome assembly.This first draft Tni-FNL genome assembly was enabled by complementary long-read technologies and represents a high-quality, well-annotated genome that provides novel insight into the complexity of this insect cell line and can serve as a reference for future large-scale genome engineering work in this and other similar recombinant protein production hosts.


April 21, 2020

Diverse Vectors and Mechanisms Spread New Delhi Metallo-ß-Lactamases among Carbapenem-Resistant Enterobacteriaceae in the Greater Boston Area.

New Delhi metallo-beta-lactamases (NDMs) are an uncommon but emerging cause of carbapenem resistance in the United States. Genomic factors promoting their domestic spread remain poorly characterized. A prospective genomic surveillance program among Boston-area hospitals identified multiple new occurrences of NDM-carrying strains of Escherichia coli and Enterobacter cloacae complex in inpatient and outpatient settings, representing the first occurrences of NDM-mediated resistance since initiating genomic surveillance in 2011. Cases included domestic patients with no international exposures. PacBio sequencing of isolates identified strain characteristics, resistance genes, and the complement of mobile vectors mediating spread. Analyses revealed a common 3,114-bp region containing the blaNDM gene, with carriage of this conserved region among unique strains by diverse transposon and plasmid backbones. Functional studies revealed a broad capacity for blaNDM transmission by conjugation, transposition, and complex interplasmid recombination events. NDMs represent a rapidly spreading form of drug resistance that can occur in inpatient and outpatient settings and in patients without international exposures. In contrast to Tn4401-based spread of Klebsiella pneumoniae carbapenemases (KPCs), diverse transposable elements mobilize NDM enzymes, commonly with other resistance genes, enabling naive strains to acquire multi- and extensively drug-resistant profiles with single transposition or plasmid conjugation events. Genomic surveillance provides effective means to rapidly identify these gene-level drivers of resistance and mobilization in order to inform clinical decisions to prevent further spread.Copyright © 2019 American Society for Microbiology.


April 21, 2020

Effector gene reshuffling involves dispensable mini-chromosomes in the wheat blast fungus.

Newly emerged wheat blast disease is a serious threat to global wheat production. Wheat blast is caused by a distinct, exceptionally diverse lineage of the fungus causing rice blast disease. Through sequencing a recent field isolate, we report a reference genome that includes seven core chromosomes and mini-chromosome sequences that harbor effector genes normally found on ends of core chromosomes in other strains. No mini-chromosomes were observed in an early field strain, and at least two from another isolate each contain different effector genes and core chromosome end sequences. The mini-chromosome is enriched in transposons occurring most frequently at core chromosome ends. Additionally, transposons in mini-chromosomes lack the characteristic signature for inactivation by repeat-induced point (RIP) mutation genome defenses. Our results, collectively, indicate that dispensable mini-chromosomes and core chromosomes undergo divergent evolutionary trajectories, and mini-chromosomes and core chromosome ends are coupled as a mobile, fast-evolving effector compartment in the wheat pathogen genome.


April 21, 2020

Comprehensive transcriptome analysis reveals genes potentially involved in isoflavone biosynthesis in Pueraria thomsonii Benth.

Pueraria thomsonii Benth is an important medicinal plant. Transcriptome sequencing, unigene assembly, the annotation of transcripts and the study of gene expression profiles play vital roles in gene function research. However, the full-length transcriptome of P. thomsonii remains unknown. Here, we obtained 44,339 nonredundant transcripts of P. thomsonii by using the PacBio RS II Isoform and Illumina sequencing platforms, of which 43,195 were annotated genes. Compared with the expression levels in the plant roots, those of transcripts with a |fold change| = 4 and FDR < 0.01 in the leaves or stems were assigned as differentially expressed transcripts (DETs). In total, we found 9,225 DETs, 32 of which came from structural genes that were potentially involved in isoflavone biosynthesis. The expression profiles of 8 structural genes from the RNA-Seq data were validated by qRT-PCR. We identified 437 transcription factors (TFs) that were positively or negatively correlated with at least 1 of the structural genes involved in isoflavone biosynthesis using Pearson correlation coefficients (r) (r > 0.8 or r < -0.8). We also identified a total of 32 microRNAs (miRNAs), which targeted 805 transcripts. These miRNAs caused enriched function in 'ATP binding', 'defense response', 'ADP binding', and 'signal transduction'. Interestingly, MIR156a potentially promoted isoflavone biosynthesis by repressing SBP, and MIR319 promoted isoflavone biosynthesis by repressing TCP and HB-HD-ZIP. Finally, we identified 2,690 alternative splicing events, including that of the structural genes of trans-cinnamate 4-monooxygenase and pullulanase, which are potentially involved in the biosynthesis of isoflavone and starch, respectively, and of three TFs potentially involved in isoflavone biosynthesis. Together, these results provide us with comprehensive insight into the gene expression and regulation of P. thomsonii.


April 21, 2020

A chromosome-level sequence assembly reveals the structure of the Arabidopsis thaliana Nd-1 genome and its gene set.

In addition to the BAC-based reference sequence of the accession Columbia-0 from the year 2000, several short read assemblies of THE plant model organism Arabidopsis thaliana were published during the last years. Also, a SMRT-based assembly of Landsberg erecta has been generated that identified translocation and inversion polymorphisms between two genotypes of the species. Here we provide a chromosome-arm level assembly of the A. thaliana accession Niederzenz-1 (AthNd-1_v2c) based on SMRT sequencing data. The best assembly comprises 69 nucleome sequences and displays a contig length of up to 16 Mbp. Compared to an earlier Illumina short read-based NGS assembly (AthNd-1_v1), a 75 fold increase in contiguity was observed for AthNd-1_v2c. To assign contig locations independent from the Col-0 gold standard reference sequence, we used genetic anchoring to generate a de novo assembly. In addition, we assembled the chondrome and plastome sequences. Detailed analyses of AthNd-1_v2c allowed reliable identification of large genomic rearrangements between A. thaliana accessions contributing to differences in the gene sets that distinguish the genotypes. One of the differences detected identified a gene that is lacking from the Col-0 gold standard sequence. This de novo assembly extends the known proportion of the A. thaliana pan-genome.


April 21, 2020

Complete Genome Sequence of “Candidatus Thioglobus sp.” Strain NP1, an Open-Ocean Isolate from the SUP05 Clade of Marine Gammaproteobacteria

Candidatus Thioglobus sp.textquotedblright strain NP1 is an open-ocean isolate from the SUP05 clade of Gammaproteobacteria. Whole-genome comparisons of strain NP1 to other sequenced isolates from the SUP05 clade indicate that it represents a new species of SUP05 that lacks the ability to fix inorganic carbon using the Calvin-Benson-Bassham cycle.


April 21, 2020

Genome Analyses of a New Mycoplasma Species from the Scorpion Centruroides vittatus.

Arthropod Mycoplasma are little known endosymbionts in insects, primarily known as plant disease vectors. Mycoplasma in other arthropods such as arachnids are unknown. We report the first complete Mycoplasma genome sequenced, identified, and annotated from a scorpion, Centruroides vittatus, and designate it as Mycoplasma vittatus We find the genome is at least a 683,827 bp single circular chromosome with a GC content of 42.7% and with 987 protein-coding genes. The putative virulence determinants include 11 genes associated with the virulence operon associated with protein synthesis or DNA transcription and ten genes with antibiotic and toxic compound resistance. Comparative analysis revealed that the M. vittatus genome is smaller than other Mycoplasma genomes and exhibits a higher GC content. Phylogenetic analysis shows M. vittatus as part of the Hominis group of Mycoplasma As arthropod genomes accumulate, further novel Mycoplasma genomes may be identified and characterized. Copyright © 2019 Yamashita et al.


April 21, 2020

Parallels between natural selection in the cold-adapted crop-wild relative Tripsacum dactyloides and artificial selection in temperate adapted maize.

Artificial selection has produced varieties of domesticated maize that thrive in temperate climates around the world. However, the direct progenitor of maize, teosinte, is indigenous only to a relatively small range of tropical and subtropical latitudes and grows poorly or not at all outside of this region. Tripsacum, a sister genus to maize and teosinte, is naturally endemic to the majority of areas in the western hemisphere where maize is cultivated. A full-length reference transcriptome for Tripsacum dactyloides generated using long-read Iso-Seq data was used to characterize independent adaptation to temperate climates in this clade. Genes related to phospholipid biosynthesis, a critical component of cold acclimation in other cold-adapted plant lineages, were enriched among those genes experiencing more rapid rates of protein sequence evolution in T. dactyloides. In contrast with previous studies of parallel selection, we find that there is a significant overlap between the genes that were targets of artificial selection during the adaptation of maize to temperate climates and those that were targets of natural selection in temperate-adapted T. dactyloides. Genes related to growth, development, response to stimulus, signaling, and organelles were enriched in the set of genes identified as both targets of natural and artificial selection. © 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.


April 21, 2020

The Reference Genome Sequence of Scutellaria baicalensis Provides Insights into the Evolution of Wogonin Biosynthesis.

Scutellaria baicalensis Georgi is important in Chinese traditional medicine where preparations of dried roots, “Huang Qin,” are used for liver and lung complaints and as complementary cancer treatments. We report a high-quality reference genome sequence for S. baicalensis where 93% of the 408.14-Mb genome has been assembled into nine pseudochromosomes with a super-N50 of 33.2 Mb. Comparison of this sequence with those of closely related species in the order Lamiales, Sesamum indicum and Salvia splendens, revealed that a specialized metabolic pathway for the synthesis of 4′-deoxyflavone bioactives evolved in the genus Scutellaria. We found that the gene encoding a specific cinnamate coenzyme A ligase likely obtained its new function following recent mutations, and that four genes encoding enzymes in the 4′-deoxyflavone pathway are present as tandem repeats in the genome of S. baicalensis. Further analyses revealed that gene duplications, segmental duplication, gene amplification, and point mutations coupled to gene neo- and subfunctionalizations were involved in the evolution of 4′-deoxyflavone synthesis in the genus Scutellaria. Our study not only provides significant insight into the evolution of specific flavone biosynthetic pathways in the mint family, Lamiaceae, but also will facilitate the development of tools for enhancing bioactive productivity by metabolic engineering in microbes or by molecular breeding in plants. The reference genome of S. baicalensis is also useful for improving the genome assemblies for other members of the mint family and offers an important foundation for decoding the synthetic pathways of bioactive compounds in medicinal plants.Copyright © 2019 The Authors. Published by Elsevier Inc. All rights reserved.


April 21, 2020

Long-read sequence and assembly of segmental duplications.

We have developed a computational method based on polyploid phasing of long sequence reads to resolve collapsed regions of segmental duplications within genome assemblies. Segmental Duplication Assembler (SDA; https://github.com/mvollger/SDA ) constructs graphs in which paralogous sequence variants define the nodes and long-read sequences provide attraction and repulsion edges, enabling the partition and assembly of long reads corresponding to distinct paralogs. We apply it to single-molecule, real-time sequence data from three human genomes and recover 33-79 megabase pairs (Mb) of duplications in which approximately half of the loci are diverged (<99.8%) compared to the reference genome. We show that the corresponding sequence is highly accurate (>99.9%) and that the diverged sequence corresponds to copy-number-variable paralogs that are absent from the human reference genome. Our method can be applied to other complex genomes to resolve the last gene-rich gaps, improve duplicate gene annotation, and better understand copy-number-variant genetic diversity at the base-pair level.


April 21, 2020

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.


April 21, 2020

Tools and Strategies for Long-Read Sequencing and De Novo Assembly of Plant Genomes.

The commercial release of third-generation sequencing technologies (TGSTs), giving long and ultra-long sequencing reads, has stimulated the development of new tools for assembling highly contiguous genome sequences with unprecedented accuracy across complex repeat regions. We survey here a wide range of emerging sequencing platforms and analytical tools for de novo assembly, provide background information for each of their steps, and discuss the spectrum of available options. Our decision tree recommends workflows for the generation of a high-quality genome assembly when used in combination with the specific needs and resources of a project.Copyright © 2019 Elsevier Ltd. All rights reserved.


April 21, 2020

Characterizing the major structural variant alleles of the human genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. Copyright © 2018 Elsevier Inc. All rights reserved.


April 21, 2020

Stout camphor tree genome fills gaps in understanding of flowering plant genome evolution.

We present reference-quality genome assembly and annotation for the stout camphor tree (Cinnamomum kanehirae (Laurales, Lauraceae)), the first sequenced member of the Magnoliidae comprising four orders (Laurales, Magnoliales, Canellales and Piperales) and over 9,000 species. Phylogenomic analysis of 13 representative seed plant genomes indicates that magnoliid and eudicot lineages share more recent common ancestry than monocots. Two whole-genome duplication events were inferred within the magnoliid lineage: one before divergence of Laurales and Magnoliales and the other within the Lauraceae. Small-scale segmental duplications and tandem duplications also contributed to innovation in the evolutionary history of Cinnamomum. For example, expansion of the terpenoid synthase gene subfamilies within the Laurales spawned the diversity of Cinnamomum monoterpenes and sesquiterpenes.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.