Long‐read sequence capture of the haemoglobin gene clusters across codfish species

AbstractCombining high-throughput sequencing with targeted sequence capture has become an attractive tool to study specific genomic regions of interest. Most studies have so far focused on the exome using short-read technology. These approaches are not designed to capture intergenic regions needed to reconstruct genomic organization, including regulatory regions and gene synteny. Here, we demonstrate the power of combining targeted sequence capture with long-read sequencing technology for comparative genomic analyses of the hemoglobin (Hb) gene clusters across eight species separated by up to 70 million years. Guided by the reference genome assembly of the Atlantic cod (Gadus morhua) together with genome information from draft assemblies of selected codfishes, we designed probes covering the two Hb gene clusters. Use of custom-made barcodes combined with PacBio RSII sequencing led to highly continuous assemblies of the LA (~100kb) and MN (~200kb) clusters, which include syntenic regions of coding and intergenic sequences. Our results revealed an overall conserved genetic organization and synteny of the Hb genes within this lineage, yet with several, lineage-specific gene duplications. Moreover, for some of the species examined, we identified amino acid substitutions at two sites in the Hbb1 gene as well as length polymorphisms in its regulatory region, which has previously been linked to temperature adaptation in Atlantic cod populations. This study highlights the use of targeted long-read capture as a versatile approach for comparative genomic studies by generation of a cross-species genomic resource elucidating the evolutionary history of the Hb gene family across the highly divergent group of codfishes.

Download Full-text

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Methods ◽

10.1016/j.ymeth.2021.02.003 ◽

2021 ◽

Author(s):

Blondal Thorarinn ◽

Gamba Cristina ◽

Jagd Lea Møller ◽

Su Ling ◽

Demirov Dimiter ◽

...

Keyword(s):

Sequence Capture ◽

Long Read

Download Full-text

Biosynthetic potential of uncultured Antarctic soil bacteria revealed through long-read metagenomic sequencing

The ISME Journal ◽

10.1038/s41396-021-01052-3 ◽

2021 ◽

Author(s):

Valentin Waschulin ◽

Chiara Borsetto ◽

Robert James ◽

Kevin K. Newsham ◽

Stefano Donadio ◽

...

Keyword(s):

Genome Mining ◽

Gene Clusters ◽

Biosynthetic Gene Cluster ◽

Full Length ◽

Metagenomic Sequencing ◽

Short Read ◽

Short Read Sequencing ◽

Rich Diversity ◽

Long Read ◽

The Rich

AbstractThe growing problem of antibiotic resistance has led to the exploration of uncultured bacteria as potential sources of new antimicrobials. PCR amplicon analyses and short-read sequencing studies of samples from different environments have reported evidence of high biosynthetic gene cluster (BGC) diversity in metagenomes, indicating their potential for producing novel and useful compounds. However, recovering full-length BGC sequences from uncultivated bacteria remains a challenge due to the technological restraints of short-read sequencing, thus making assessment of BGC diversity difficult. Here, long-read sequencing and genome mining were used to recover >1400 mostly full-length BGCs that demonstrate the rich diversity of BGCs from uncultivated lineages present in soil from Mars Oasis, Antarctica. A large number of highly divergent BGCs were not only found in the phyla Acidobacteriota, Verrucomicrobiota and Gemmatimonadota but also in the actinobacterial classes Acidimicrobiia and Thermoleophilia and the gammaproteobacterial order UBA7966. The latter furthermore contained a potential novel family of RiPPs. Our findings underline the biosynthetic potential of underexplored phyla as well as unexplored lineages within seemingly well-studied producer phyla. They also showcase long-read metagenomic sequencing as a promising way to access the untapped genetic reservoir of specialised metabolite gene clusters of the uncultured majority of microbes.

Download Full-text

Metagenomic exploration of the marine sponge mycale hentscheli uncovers multiple polyketide-producing bacterial symbionts

10.26686/wgtn.12444086 ◽

2020 ◽

Author(s):

Mathew Storey ◽

SK Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Defensive Symbiosis ◽

Long Read ◽

Comprehensive Picture ◽

Mycale Hentscheli

© 2020 Storey et al. Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metag-enomic investigation of Mycale hentscheli, a chemically gifted marine sponge that pos-sesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short-and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemo-types. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holo-biont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These re-sults suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small mol-ecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing mi-crobe. We also find that the microbiome of M. hentscheli is stably maintained among in-dividuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

Download Full-text

Dynamics in Secondary Metabolite Gene Clusters in Otherwise Highly Syntenic and Stable Genomes in the Fungal Genus Botrytis

Genome Biology and Evolution ◽

10.1093/gbe/evaa218 ◽

2020 ◽

Vol 12 (12) ◽

pp. 2491-2507

Author(s):

Claudio A Valero-Jiménez ◽

Maikel B F Steentjes ◽

Jason C Slot ◽

Xiaoqian Shi-Kunne ◽

Olga E Scholten ◽

...

Keyword(s):

Secondary Metabolite ◽

Gene Clusters ◽

Single Species ◽

White Rot ◽

White Rot Fungus ◽

Broad Host Range ◽

Allium Species ◽

Sclerotium Cepivorum ◽

Single Host ◽

Long Read

Abstract Fungi of the genus Botrytis infect >1,400 plant species and cause losses in many crops. Besides the broad host range pathogen Botrytis cinerea, most other species are restricted to a single host. Long-read technology was used to sequence genomes of eight Botrytis species, mostly pathogenic on Allium species, and the related onion white rot fungus, Sclerotium cepivorum. Most assemblies contained <100 contigs, with the Botrytis aclada genome assembled in 16 gapless chromosomes. The core genome and pan-genome of 16 Botrytis species were defined and the secretome, effector, and secondary metabolite repertoires analyzed. Among those genes, none is shared among all Allium pathogens and absent from non-Allium pathogens. The genome of each of the Allium pathogens contains 8–39 predicted effector genes that are unique for that single species, none stood out as potential determinant for host specificity. Chromosome configurations of common ancestors of the genus Botrytis and family Sclerotiniaceae were reconstructed. The genomes of B. cinerea and B. aclada were highly syntenic with only 19 rearrangements between them. Genomes of Allium pathogens were compared with ten other Botrytis species (nonpathogenic on Allium) and with 25 Leotiomycetes for their repertoire of secondary metabolite gene clusters. The pattern was complex, with several clusters displaying patchy distribution. Two clusters involved in the synthesis of phytotoxic metabolites are at distinct genomic locations in different Botrytis species. We provide evidence that the clusters for botcinic acid production in B. cinerea and Botrytis sinoallii were acquired by horizontal transfer from taxa within the same genus.

Download Full-text

Long-read metagenomics of soil communities reveals phylum-specific secondary metabolite dynamics

10.1101/2021.01.23.426502 ◽

2021 ◽

Author(s):

Marc W. Van Goethem ◽

Andrew R. Osborn ◽

Benjamin P. Bowen ◽

Peter F. Andeer ◽

Tami L. Swenson ◽

...

Keyword(s):

Secondary Metabolites ◽

Temporal Dynamics ◽

Functional Characterization ◽

Gene Clusters ◽

Metagenomic Sequencing ◽

Pronounced Increase ◽

Environmental Processes ◽

Soil Communities ◽

Functional Dynamics ◽

Long Read

AbstractMicrobial biosynthetic gene clusters (BGCs) encoding secondary metabolites are thought to impact a plethora of biologically mediated environmental processes, yet their discovery and functional characterization in natural microbiomes remains challenging. Here we describe deep long-read sequencing and assembly of metagenomes from biological soil crusts, a group of soil communities that are rich in BGCs. Taking advantage of the unusually long assemblies produced by this approach, we recovered nearly 3,000 BGCs for analysis, including 695 novel, full-length BGCs. Functional exploration through metatranscriptome analysis of a 3-day wetting experiment uncovered phylum-specific BGC expression upon activation from dormancy, elucidating distinct roles and complex phylogenetic and temporal dynamics in wetting processes. For example, a pronounced increase in BGC transcription occurs at night in cyanobacteria but not in other phyla, implicating BGCs in nutrient scavenging roles and niche competition. Taken together, our results demonstrate that long-read metagenomic sequencing combined with metatranscriptomic analysis provides a direct view into the functional dynamics of BGCs in environmental processes and suggests a central role of secondary metabolites in maintaining phylogenetically conserved niches within biocrusts.

Download Full-text

Metagenomic Exploration of the Marine Sponge Mycale hentscheli Uncovers Multiple Polyketide-Producing Bacterial Symbionts

mBio ◽

10.1128/mbio.02997-19 ◽

2020 ◽

Vol 11 (2) ◽

Cited By ~ 5

Author(s):

Mathew A. Storey ◽

Sarah K. Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert A. Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Content Type ◽

Defensive Symbiosis ◽

Long Read ◽

Mycale Hentscheli

ABSTRACT Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metagenomic investigation of Mycale hentscheli, a chemically gifted marine sponge that possesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short- and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemotypes. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holobiont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These results suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small molecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing microbe. We also find that the microbiome of M. hentscheli is stably maintained among individuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

Download Full-text

Metagenomic exploration of the marine sponge mycale hentscheli uncovers multiple polyketide-producing bacterial symbionts

10.26686/wgtn.12444086.v1 ◽

2020 ◽

Author(s):

Mathew Storey ◽

SK Andreassend ◽

Joe Bracegirdle ◽

Alistair Brown ◽

Robert Keyzers ◽

...

Keyword(s):

Marine Sponge ◽

Bacterial Species ◽

Gene Clusters ◽

Taxonomic Diversity ◽

Marine Sponges ◽

Metagenomic Sequencing ◽

Defensive Symbiosis ◽

Long Read ◽

Comprehensive Picture ◽

Mycale Hentscheli

© 2020 Storey et al. Marine sponges have been a prolific source of unique bioactive compounds that are presumed to act as a deterrent to predation. Many of these compounds have potential therapeutic applications; however, the lack of efficient and sustainable synthetic routes frequently limits clinical development. Here, we describe a metag-enomic investigation of Mycale hentscheli, a chemically gifted marine sponge that pos-sesses multiple distinct chemotypes. We applied shotgun metagenomic sequencing, hybrid assembly of short-and long-read data, and metagenomic binning to obtain a comprehensive picture of the microbiome of five specimens, spanning three chemo-types. Our data revealed multiple producing species, each having relatively modest secondary metabolomes, that contribute collectively to the chemical arsenal of the holo-biont. We assembled complete genomes for multiple new genera, including two species that produce the cytotoxic polyketides pateamine and mycalamide, as well as a third high-abundance symbiont harboring a proteusin-type biosynthetic pathway that appears to encode a new polytheonamide-like compound. We also identified an additional 188 biosynthetic gene clusters, including a pathway for biosynthesis of peloruside. These re-sults suggest that multiple species cooperatively contribute to defensive symbiosis in M. hentscheli and reveal that the taxonomic diversity of secondary-metabolite-producing sponge symbionts is larger and richer than previously recognized. IMPORTANCE Mycale hentscheli is a marine sponge that is rich in bioactive small mol-ecules. Here, we use direct metagenomic sequencing to elucidate highly complete and contiguous genomes for the major symbiotic bacteria of this sponge. We identify complete biosynthetic pathways for the three potent cytotoxic polyketides which have previously been isolated from M. hentscheli. Remarkably, and in contrast to previous studies of marine sponges, we attribute each of these metabolites to a different producing mi-crobe. We also find that the microbiome of M. hentscheli is stably maintained among in-dividuals, even over long periods of time. Collectively, our data suggest a cooperative mode of defensive symbiosis in which multiple symbiotic bacterial species cooperatively contribute to the defensive chemical arsenal of the holobiont.

Download Full-text

P4054 Resolving misassembled cattle immune gene clusters with hierarchical, long read sequencing

Journal of Animal Science ◽

10.2527/jas2016.94supplement4105a ◽

2016 ◽

Vol 94 (suppl_4) ◽

pp. 105-106

Author(s):

D. Bickhart ◽

J. A. Hammond ◽

J. C. Schwartz ◽

D. Harrison ◽

T. P. L. Smith

Keyword(s):

Gene Clusters ◽

Immune Gene ◽

Long Read

Download Full-text

Non-Transgenic CRISPR-Mediated Knockout of Entire Ergot Alkaloid Gene Clusters in Slow-Growing Asexual Polyploid Fungi

Toxins ◽

10.3390/toxins13020153 ◽

2021 ◽

Vol 13 (2) ◽

pp. 153

Author(s):

Simona Florea ◽

Jolanta Jaromczyk ◽

Christopher L. Schardl

Keyword(s):

Ergot Alkaloid ◽

Gene Clusters ◽

Triploid Hybrid ◽

Alkaloid Biosynthesis ◽

Cool Season ◽

Hybrid Species ◽

Guide Rnas ◽

Cas9 Nuclease ◽

Long Read ◽

Slow Growing

The Epichloë species of fungi include seed-borne symbionts (endophytes) of cool-season grasses that enhance plant fitness, although some also produce alkaloids that are toxic to livestock. Selected or mutated toxin-free endophytes can be introduced into forage cultivars for improved livestock performance. Long-read genome sequencing revealed clusters of ergot alkaloid biosynthesis (EAS) genes in Epichloë coenophiala strain e19 from tall fescue (Lolium arundinaceum) and Epichloë hybrida Lp1 from perennial ryegrass (Lolium perenne). The two homeologous clusters in E. coenophiala—a triploid hybrid species—were 196 kb (EAS1) and 75 kb (EAS2), and the E. hybrida EAS cluster was 83 kb. As a CRISPR-based approach to target these clusters, the fungi were transformed with ribonucleoprotein (RNP) complexes of modified Cas9 nuclease (Cas9-2NLS) and pairs of single guide RNAs (sgRNAs), plus a transiently selected plasmid. In E. coenophiala, the procedure generated deletions of EAS1 and EAS2 separately, as well as both clusters simultaneously. The technique also gave deletions of the EAS cluster in E. hybrida and of individual alkaloid biosynthesis genes (dmaW and lolC) that had previously proved difficult to delete in E. coenophiala. Thus, this facile CRISPR RNP approach readily generates non-transgenic endophytes without toxin genes for use in research and forage cultivar improvement.

Download Full-text