scholarly journals Optimised DNA extraction and library preparation for minute arthropods: application to target enrichment in chalcid wasps used for biocontrol

2018 ◽  
Author(s):  
Astrid Cruaud ◽  
Sabine Nidelet ◽  
Pierre Arnal ◽  
Audrey Weber ◽  
Lucian Fusu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Evolutionary History ◽  
Animal Species ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Target Enrichment ◽  
Library Preparation ◽  
Single Person ◽  
History Of ◽  
Set Up

AbstractEnriching subsets of the genome prior to sequencing allows focusing effort on regions that are relevant to answer specific questions. As experimental design can be adapted to sequence many samples simultaneously, using such approach also contributes to reduce cost. In the field of ecology and evolution, target enrichment is increasingly used for genotyping of plant and animal species or to better understand the evolutionary history of important lineages through the inference of statistically robust phylogenies. Limitations to routine target enrichment by research laboratories are both the complexity of current protocols and low input DNA quantity. Thus, working with tiny organisms such as micro-arthropods can be challenging. Here, we propose easy to set up optimisations for DNA extraction and library preparation prior to target enrichment. Prepared libraries were used to capture 1432 Ultra-Conserved Elements (UCEs) from microhymenoptera (Chalcidoidea), which are among the tiniest insects on Earth and the most commercialized worldwide for biological control purposes. Results show no correlation between input DNA quantities (1.8-250ng, 0.4 ng with an extra whole genome amplification step) and the number of sequenced UCEs. Phylogenetic inferences highlight the potential of UCEs to solve relationships within the families of chalcid wasps, which has not been achieved so far. The protocol (library preparation + target enrichment), allows processing 96 specimens in five working days, by a single person, without requiring the use of expensive robotic molecular biology platforms, which could help to generalize the use of target enrichment for minute specimens.

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

scholarly journals Culicidae evolutionary history focusing on the Culicinae subfamily based on mitochondrial phylogenomics

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Alexandre Freitas da Silva ◽  
Laís Ceschini Machado ◽  
Marcia Bicudo de Paula ◽  
Carla Júlia da Silva Pessoa Vieira ◽  
Roberta Vieira de Morais Bronzoni ◽  
...  
Keyword(s):  
Data Mining ◽  
Evolutionary History ◽  
Mosquito Species ◽  
Whole Genome ◽  
Lack Of Information ◽  
Fundamental Information ◽  
History Of ◽  
Medical Importance ◽  
Low Coverage

Abstract Mosquitoes are insects of medical importance due their role as vectors of different pathogens to humans. There is a lack of information about the evolutionary history and phylogenetic positioning of the majority of mosquito species. Here we characterized the mitogenomes of mosquito species through low-coverage whole genome sequencing and data mining. A total of 37 draft mitogenomes of different species were assembled from which 16 are newly-sequenced species. We datamined additional 49 mosquito mitogenomes, and together with our 37 mitogenomes, we reconstructed the evolutionary history of 86 species including representatives from 15 genera and 7 tribes. Our results showed that most of the species clustered in clades with other members of their own genus with exception of Aedes genus which was paraphyletic. We confirmed the monophyletic status of the Mansoniini tribe including both Coquillettidia and Mansonia genus. The Aedeomyiini and Uranotaeniini were consistently recovered as basal to other tribes in the subfamily Culicinae, although the exact relationships among these tribes differed between analyses. These results demonstrate that low-coverage sequencing is effective to recover mitogenomes, establish phylogenetic knowledge and hence generate basic fundamental information that will help in the understanding of the role of these species as pathogen vectors.

scholarly journals Evolutionary history of Tibetans inferred from whole-genome sequencing

PLoS Genetics ◽  
2017 ◽  
Vol 13 (4) ◽  
pp. e1006675 ◽  
Author(s):  
Hao Hu ◽  
Nayia Petousi ◽  
Gustavo Glusman ◽  
Yao Yu ◽  
Ryan Bohlender ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Evolutionary History ◽  
Whole Genome ◽  
History Of

scholarly journals Cereulide synthetase acquisition and loss events within the evolutionary history of Group III Bacillus cereus sensu lato facilitate the transition between emetic and diarrheal foodborne pathogen

2020 ◽  
Author(s):  
Laura M. Carroll ◽  
Martin Wiedmann
Keyword(s):  
Bacillus Cereus ◽  
Evolutionary History ◽  
Common Ancestor ◽  
Reference Genome ◽  
Foodborne Pathogen ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Group Iii ◽  
History Of ◽  
Bacillus Cereus Sensu Lato

AbstractCereulide-producing members of Bacillus cereus sensu lato (B. cereus s.l.) Group III, also known as “emetic B. cereus”, possess cereulide synthetase, a plasmid-encoded, non-ribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by “emetic B. cereus” has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize Group III B. cereus s.l. genomes which possess ces (ces-positive) alongside their closely related ces-negative counterparts to (i) assess the genomic diversity encompassed by “emetic B. cereus”, and (ii) identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive Group III B. cereus s.l. genomes and the ces-negative genomes interspersed among them (n = 150), we show that “emetic B. cereus” is not clonal; rather, multiple lineages within Group III harbor cereulide-producing strains, all of which share a common ancestor incapable of producing cereulide (posterior probability [PP] 0.86-0.89). The ST 26 common ancestor was predicted to have emerged as ces-negative (PP 0.60-0.93) circa 1904 (95% highest posterior density [HPD] interval 1837.1-1957.8) and first acquired the ability to produce cereulide before 1931 (95% HPD 1893.2-1959.0). Three subsequent ces loss events within ST 26 were observed, including among isolates responsible for B. cereus s.l. toxicoinfection (i.e., “diarrheal” illness).Importance“B. cereus” is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., “emetic” syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., “diarrheal” syndrome). Here, we show that “emetic B. cereus” is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some Group III B. cereus s.l. populations to oscillate between diarrheal and emetic foodborne pathogen over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus s.l. outbreaks, as some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.

scholarly journals Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae

mBio ◽  
2018 ◽  
Vol 9 (1) ◽  
Author(s):  
Pierre Gladieux ◽  
Bradford Condon ◽  
Sebastien Ravel ◽  
Darren Soanes ◽  
Joao Leodato Nunes Maciel ◽  
...  
Keyword(s):  
Gene Flow ◽  
South America ◽  
Magnaporthe Oryzae ◽  
Rice Blast ◽  
Evolutionary History ◽  
Blast Disease ◽  
Whole Genome ◽  
Disease Emergence ◽  
Wheat Blast ◽  
History Of

ABSTRACT Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae. These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.

scholarly journals A DNA extraction protocol for improved DNA yield from individual mosquitoes

F1000Research ◽  
2015 ◽  
Vol 4 ◽  
pp. 1314 ◽  
Author(s):  
Catelyn C. Nieman ◽  
Youki Yamasaki ◽  
Travis C. Collier ◽  
Yoosook Lee
Keyword(s):  
Next Generation Sequencing ◽  
Dna Extraction ◽  
Whole Genome Amplification ◽  
Whole Genome ◽  
Single Individual ◽  
Dna Amount ◽  
Dna Yield ◽  
Extraction Protocol ◽  
Genome Representation ◽  
Generation Sequencing

Typical DNA extraction protocols from commercially available kits provide an adequate amount of DNA from a single individual mosquito sufficient for PCR-based assays. However, next-generation sequencing applications and high-throughput SNP genotyping assays exposed the limitation of DNA quantity one usually gets from a single individual mosquito. Whole genome amplification could alleviate the issue but it also creates bias in genome representation. While trying to find alternative DNA extraction protocols for improved DNA yield, we found that a combination of the tissue lysis protocol from Life Technologies and the DNA extraction protocol from Qiagen yielded a higher DNA amount than the protocol using the Qiagen or Life Technologies kit only. We have not rigorously tested all the possible combinations of extraction protocols; we also only tested this on mosquito samples. Therefore, our finding should be noted as a suggestion for improving people’s own DNA extraction protocols and not as an advertisement of a commercially available product.

scholarly journals Cereulide Synthetase Acquisition and Loss Events within the Evolutionary History of Group III Bacillus cereus Sensu Lato Facilitate the Transition between Emetic and Diarrheal Foodborne Pathogens

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Laura M. Carroll ◽  
Martin Wiedmann
Keyword(s):  
Bacillus Cereus ◽  
Foodborne Pathogens ◽  
Evolutionary History ◽  
Reference Genome ◽  
Genomic Diversity ◽  
Whole Genome ◽  
Content Type ◽  
Group Iii ◽  
History Of ◽  
Highest Posterior Density

ABSTRACT Cereulide-producing members of Bacillus cereus sensu lato group III (also known as emetic B. cereus) possess cereulide synthetase, a plasmid-encoded, nonribosomal peptide synthetase encoded by the ces gene cluster. Despite the documented risks that cereulide-producing strains pose to public health, the level of genomic diversity encompassed by emetic B. cereus has never been evaluated at a whole-genome scale. Here, we employ a phylogenomic approach to characterize group III B. cereus sensu lato genomes which possess ces (ces positive) alongside their closely related, ces-negative counterparts (i) to assess the genomic diversity encompassed by emetic B. cereus and (ii) to identify potential ces loss and/or gain events within the evolutionary history of the high-risk and medically relevant sequence type (ST) 26 lineage often associated with emetic foodborne illness. Using all publicly available ces-positive group III B. cereus sensu lato genomes and the ces-negative genomes interspersed among them (n = 159), we show that emetic B. cereus is not clonal; rather, multiple lineages within group III harbor cereulide-producing strains, all of which share an ancestor incapable of producing cereulide (posterior probability = 0.86 to 0.89). Members of ST 26 share an ancestor that existed circa 1748 (95% highest posterior density [HPD] interval = 1246.89 to 1915.64) and first acquired the ability to produce cereulide before 1876 (95% HPD = 1641.43 to 1946.70). Within ST 26 alone, two subsequent ces gain events were observed, as well as three ces loss events, including among isolates responsible for B. cereus sensu lato toxicoinfection (i.e., “diarrheal” illness). IMPORTANCE B. cereus is responsible for thousands of cases of foodborne disease each year worldwide, causing two distinct forms of illness: (i) intoxication via cereulide (i.e., emetic syndrome) or (ii) toxicoinfection via multiple enterotoxins (i.e., diarrheal syndrome). Here, we show that emetic B. cereus is not a clonal, homogenous unit that resulted from a single cereulide synthetase gain event followed by subsequent proliferation; rather, cereulide synthetase acquisition and loss is a dynamic, ongoing process that occurs across lineages, allowing some group III B. cereus sensu lato populations to oscillate between diarrheal and emetic foodborne pathogens over the course of their evolutionary histories. We also highlight the care that must be taken when selecting a reference genome for whole-genome sequencing-based investigation of emetic B. cereus sensu lato outbreaks, since some reference genome selections can lead to a confounding loss of resolution and potentially hinder epidemiological investigations.

scholarly journals Optimized DNA extraction and library preparation for minute arthropods: Application to target enrichment in chalcid wasps used for biocontrol

2019 ◽  
Vol 19 (3) ◽  
pp. 702-710 ◽  
Author(s):  
Astrid Cruaud ◽  
Sabine Nidelet ◽  
Pierre Arnal ◽  
Audrey Weber ◽  
Lucian Fusu ◽  
...  
Keyword(s):  
Dna Extraction ◽  
Target Enrichment ◽  
Library Preparation

scholarly journals Migration without interbreeding: evolutionary history of a highly selfing Mediterranean grass inferred from whole genomes

2020 ◽  
Author(s):  
Christoph Stritt ◽  
Elena L. Gimmi ◽  
Michele Wyler ◽  
Abdelmonaim H. Bakali ◽  
Aleksandra Skalska ◽  
...  
Keyword(s):  
Evolutionary History ◽  
Brachypodium Distachyon ◽  
Genotypic Diversity ◽  
Plant Evolution ◽  
Whole Genome ◽  
Continental Scale ◽  
Whole Genomes ◽  
History Of ◽  
Adaptive Phenotypic Plasticity ◽  
Independent Expansion

AbstractWhole genome sequences and coalescence theory allow the study of plant evolution in unprecedented detail. In this study we extend the genomic resources for the wild Mediterranean grass Brachypodium distachyon to investigate the scale of population structure and its underlying history at whole-genome resolution. The analysis of 196 accessions, spanning the Mediterranean from Iberia to Iraq, shows that the interplay of high selfing and seed dispersal rates has shaped genetic structure. At the continental scale, evolution in B. distachyon is characterized by the independent expansion of three lineages during the Upper Pleistocene. Today, these lineages may occur in sympatry yet do not interbreed. At the local scale, dispersal and selfing interact to maintain high genotypic diversity. Our study lays a foundation for the study of microevolution in B. distachyon and identifies adaptive phenotypic plasticity and frequency-dependent selection as key themes to be addressed with this model system.

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