Biology subjects

Haverkamp, T. H. A.

Publications and source records attributed to Haverkamp, T. H. A..

9 recordsLinked to original sources

bioRxiv2024

DNA Extraction Protocols for Animal Fecal Material on Blood Spot Cards

BackgroundCollecting fecal samples using dry preservatives is an attractive option in large epidemiological studies as they are easy to use, cheap and independent of cold chain logistics. Here, we test four DNA extraction methods with the aim of identifying an efficient procedure to extract high-quality DNA from fecal material of canine, sheep, equine, bovine, and pig collected on dry blood spot cards, with the goal of generating good quality shotgun metagenomics datasets. Further, the suitability of Illumina shotgun metagenomic sequencing at 20million PE read depth was assessed on its ability to successfully characterize the taxonomic and functional aspects of the resulting fecal microbiome. MethodsDNA was extracted from pig feces and mock communities collected on blood spot cards using four DNA extraction methods; two different methods of the QIAsymphony(R) PowerFecal(R) Pro DNA Kit, the ZymoBIOMICS DNA Miniprep Kit, and the MagNA Pure 96 DNA and Viral NA Small Volume Kit. Possible extraction bias was controlled by amplicon sequencing of mock communities. Fecal samples from canine, sheep, equine, bovine, and pig were thereafter subjected to the best performing DNA extraction method and shotgun metagenomic sequencing to determine sequencing efforts for functional and taxonomic analysis. ResultsThe four DNA extraction methods demonstrated similar community composition in the sequenced bacterial mock community. The QIAsymphony(R) PowerFecal(R) Pro DNA Kit was identified as the DNA extraction method of choice, and the resulting DNA was subjected to shotgun metagenomic sequencing with 20million PE reads. We found that higher number of reads increased the richness of observed genera between 100,000 and 5 million reads, after which higher sequencing effort did not increase the richness of the metagenomes. As for functional analysis, the number of low abundance functions in the metagenomes of the animals feces increased with sequencing depth above 20 million PE reads. ConclusionOur experiments identified several methods suitable for extracting DNA from feces collected on blood spot cards. The Qiagens Blood and Tissue kit on the QiaSymphony platform fulfilled the criteria of high yield, quality, and unbiased DNA, while maintaining high throughput for shotgun metagenomic sequencing. A sequencing depth of 20 million PE reads proved adequate for taxonomic estimations and identifying common functional pathways. Detecting rarer traits, however, requires more sequencing effort.

microbiology↗

bioRxiv2021

Detection of Campylobacter in air samples from poultry houses using shot-gun metagenomics - a pilot study

BackgroundFoodborne pathogens such as Campylobacter jejuni are responsible for a large fraction of the gastrointestinal infections worldwide associated with poultry meat. Campylobacter spp. can be found in the chicken fecal microbiome and can contaminate poultry meat during the slaughter process. The current standard methods to detect these pathogens at poultry farms use fecal dropping or boot swaps in combination with cultivation / PCR. In this study, we have used air filters in combination with shotgun metagenomics for the detection of Campylobacter in poultry houses and MOCK communities to test the applicability of this approach for the detection of foodborne pathogens. ResultsThe spiked MOCK communities showed that we could detect as little as 200 CFU Campylobacter per sample using our protocols. Since we were interested in detecting Campylobacter, a DNA extraction protocol for Gram negative bacteria was chosen, and as expected, we found that the DNA extraction protocol created a substantial bias affecting the community composition of the MOCK communities. It can be expected that the same bias is present for poultry house samples analyzed. We observed significant amounts of Campylobacter on the air filters using both real-time PCR as well as shotgun metagenomics, irrespective of the amount of spiked in Campylobacter cells, suggesting that the flocks in both houses harboured Campylobacter spp.. Interestingly, in both houses we find diverse microbial communities present in the indoor air. In addition, have we tested the Campylobacter detection rate using shotgun metagenomics by spiking with different levels of C. jejuni cells in both the mock and the house samples. This showed that even with limited sequencing Campylobacter is detectable in samples with low abundance. ConclusionsThese results show that air sampling of poultry houses in combination with shotgun metagenomics can detect and identify Campylobacter spp. present at low levels. This is important since early detection of Campylobacter in food production can help to decrease the number of food-borne infections.

microbiology↗

bioRxiv2020

Viruses of the Thermotogota; the vehicles on the highways of gene sharing.

Phylogenomic analyses of bacteria from the phylum Thermotogota have shown extensive lateral gene transfer (LGT) with distantly related organisms, particularly with Firmicutes. One likely mechanism of such DNA transfer is viruses. However, to date only three temperate viruses have been characterized in this phylum, all infecting bacteria from the Marinitoga genus. Here we report 17 proviruses integrated into genomes of eight Thermotogota genera and induce viral particle production from one of the proviruses. The proviruses fall into two groups based on sequence similarity, gene synteny and taxonomic classification. Proviruses of one group are found in six genera and are similar to the previously identified Marinitoga viruses, while proviruses from the second group are only distantly related to the proviruses of the first group, have different genome organization and are found in only two genera. Both groups are closely related to Firmicutes in genomic and phylogenetic analyses, and one of the groups show evidence of very recent LGT and are therefore likely capable of infecting cells from both phyla. We conjecture that viruses are responsible for a large portion of the observed gene flow between Firmicutes and Thermotogota.

evolutionary biology↗

bioRxiv2019

Metagenomic shotgun analyses reveal complex patterns of intra- and interspecific variation in the intestinal microbiomes of codfishes

The relative importance of host-specific selection or environmental factors in determining the composition of the intestinal microbiome in wild vertebrates remains poorly understood. Here, we use metagenomic shotgun sequencing of individual specimens to compare the intra- and interspecific variation of intestinal microbiome communities in two ecotypes (NEAC and NCC) of Atlantic cod (Gadus morhua) - that have distinct behavior and habitats- and three Gadidae species that occupy a range of ecological niches. Interestingly, we find significantly diverged microbiomes amongst the two Atlantic cod ecotypes. Interspecific patterns of variation are more variable, with significantly diverged communities for most species comparisons, apart from the comparison between coastal cod (NCC) and Norway pout (Trisopterus esmarkii), whose community compositions are not significantly diverged. The absence of consistent species-specific microbiomes suggests that external environmental factors, such as temperature, diet or a combination there-off comprise major drivers of the intestinal community composition of codfishes. ImportanceThe composition of the intestinal microbial community associated with teleost fish is influenced by a diversity of factors, ranging from internal factors (such as host-specific selection) to external factors (such as niche occupation). These factors are often difficult to separate, as differences in niche occupation (e.g. diet, temperature or salinity) may correlate with distinct evolutionary trajectories. Here, we investigate four gadoid species with contrasting levels of evolutionary separation and niche occupation. Using metagenomic shotgun sequencing, we observe distinct microbiomes amongst two Atlantic cod (Gadus morhua) ecotypes (NEAC and NCC) with distinct behavior and habitats. In contrast, interspecific patterns of variation are more variable. For instance, we do not observe interspecific differentiation between the microbiomes of coastal cod (NCC) and Norway pout (Trisopterus esmarkii) whose lineages have evolutionary separated over 20 million years ago. The observed pattern of microbiome variation in these gadoid species is therefore most parsimoniously explained by differences in niche occupation.

microbiology↗

bioRxiv2019

Switching on the light: using metagenomic shotgun sequencing to characterize the intestinal microbiome of Atlantic cod

The biological roles of the intestinal microbiome and how it is impacted by environmental factors are yet to be determined in wild marine fish species. Atlantic cod (Gadus morhua) is an ecologically important species with a wide-spread distribution in the North Atlantic Ocean. 16S rRNA-based amplicon analyses found no geographical differentiation between the intestinal microbiome of Atlantic cod from different locations. Nevertheless, it is unclear if this lack of differentiation results from an insufficient resolution of this method to resolve fine-scaled biological complexity. Here, we take advantage of the increased resolution provided by metagenomic shotgun sequencing to investigate the intestinal microbiome of 19 adult Atlantic cod individuals from two coastal populations in Norway - located 470 km apart. Our results show that the intestinal microbiome is dominated by the Vibrionales order, consisting of varying abundances of Photobacterium, Aliivibrio and Vibrio species. Moreover, resolving the species community to unprecedented resolution, we identify two abundant species, P. iliopiscarium and P. kishitanii, which comprise over 50% of the classified reads. Interestingly, genomic data shows that the intestinal P. kishitanii strains have functionally intact lux genes, and its high abundance suggests that fish intestines form an important part of its ecological niche. These observations support a hypothesis that bioluminescence plays an ecological role in the marine food web. Despite our improved taxonomical resolution, we identify no geographical differences in bacterial community structure, indicating that the intestinal microbiome of these coastal cod is colonized by a limited number of closely related bacterial species with a broad geographical distribution that are well suited to thrive in this host-associated environment.

microbiology↗

bioRxiv2018

Thermosipho spp. immune system differences affect variation in genome size and geographical distributions

Thermosipho species inhabit thermal environments such as marine hydrothermal vents, petroleum reservoirs and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents and hotsprings. Comparative genomics of 15 Thermosipho genomes separated them into three distinct species with different habitat distributions: the widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. Moreover, the species can be differentiated on the basis of genome size, genome content and immune system composition. For instance, the T. africanus genomes are largest and contained the most carbohydrate metabolism genes, which could explain why these isolates were obtained from ecologically more divergent habitats. Nonetheless, all the Thermosipho genomes, like other Thermotogae genomes, show evidence of genome streamlining. Genome size differences between the species could further be correlated to differences in defense capacities against foreign DNA, which influence recombination via HGT. The smallest genomes are found in T. affectus that contain both CRISPR-cas Type I and III systems, but no RM system genes. We suggest that this has caused these genomes to be almost devoid of mobile elements, contrasting the two other species genomes that contain a higher abundance of mobile elements combined with different immune system configurations. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA.

microbiology↗

bioRxiv2018

Genomic insights into metabolism and phylogeography of the mesophilic Thermotogae genus Mesotoga

The genus Mesotoga, the only described mesophilic Thermotogae lineage, is common in mesothermic anaerobic hydrocarbon-rich environments. Besides mesophily, Mesotoga displays lineage-specific phenotypes, such as no or little H2 production and dependence on sulfur-compound reduction, which may influence its ecological role. We used comparative genomics of 18 Mesotoga strains (pairwise 16S rRNA identity > 99%) and a transcriptome of M. prima to investigate how life at moderate temperatures affects phylogeography and to interrogate the genomic features of its lineage-specific metabolism. We propose that Mesotoga accomplish H2 oxidation and thiosulfate reduction using a sulfide dehydrogenase and a hydrogenase-complex and that a pyruvate:ferredoxin oxidoreductase acquired from Clostridia is responsible for oxidizing acetate. Phylogenetic analysis revealed three distinct Mesotoga lineages (89.6-99.9% average nucleotide identity [ANI] within lineages, 79.3-87.6% ANI between lineages) having different geographic distribution patterns and high levels of intra-lineage recombination but little geneflow between lineages. Including data from metagenomes, phylogeographic patterns suggest that geographical separation historically has been more important for Mesotoga than hyperthermophilic Thermotoga and we hypothesize that distribution of Mesotoga is constrained by their anaerobic lifestyle. Our data also suggest that recent anthropogenic activities and environments (e.g., wastewater treatment, oil exploration) have expanded Mesotoga habitats and dispersal capabilities.\n\nOriginality-Significance StatementThis study comprises one of the first whole-genome-based phylogeographic analyses of anaerobic mesophiles, and our data suggest that such microbes are more restricted by geography than are thermophiles (and mesophilic aerobes). This is likely to be a general trait for similar anaerobic organisms - and therefore broadly relevant to and testable in other environments. Moreover, Mesotoga bacteria are part of the largely understudied subsurface ecosystem that has relatively recently been recognized as a new and important biosphere. Understanding the forces responsible for the distribution of organisms in the subsurface, as well as the identification of genes responsible for Mesotogas distinct metabolism, will contribute to the understanding of these communities.

evolutionary biology↗

bioRxiv2017

A single Vibrionales 16S rRNA oligotype dominates the intestinal microbiome in two geographically separated Atlantic cod populations

BackgroundHost-microbe interactions are particularly intriguing in Atlantic cod (Gadus morhua), as it lacks the MHC II complex involved in presentation of extracellular pathogens. Nonetheless, little is known about the diversity of its microbiome in natural populations. Here, we use 16S rRNA high-throughput sequencing to investigate the microbial community composition in gut content and mucosa of 22 adult individuals from two coastal populations in Norway, located 470 km apart.\n\nResultsWe identify a core microbiome of 23 OTUs (97% sequence similarity) in all individuals that comprises 93% of the total number of reads. The most abundant orders are classified as Vibrionales, Fusobacteriales, Clostridiales and Bacteroidales. While mucosal samples show significantly lower diversity than gut content samples, no differences in OTU community composition are observed between the two populations. The differential abundance of oligotypes within two common OTUs does reveal limited spatial segregation. Remarkably, the most abundant OTU consists of a single oligotype (order Vibrionales, genus Photobacterium) that represents nearly 50% of the reads in both locations.\n\nConclusionsOur results show that the intestinal bacterial community of two geographically separated coastal populations of Atlantic cod is dominated by a limited number of highly abundant 16S rRNA oligotypes shared by all specimens examined. The ubiquity of these oligotypes suggests that the northern coastal Atlantic cod gut microbiome is colonized by a limited number of species with excellent dispersal capabilities that are well suited to thrive in their host environment.

microbiology↗

bioRxiv2017

Habitat generalists or specialists, insights from comparative genomic analyses of Thermosipho lineages

Thermosipho species inhabit various extreme environments such as marine hydrothermal vents, petroleum reservoirs and terrestrial hot springs. A 16S rRNA phylogeny of available Thermosipho spp. sequences suggested habitat specialists adapted to living in hydrothermal vents only, and habitat generalists inhabiting oil reservoirs, hydrothermal vents and hotsprings. Comparative genomics and recombination analysis of the genomes of 15 Thermosipho isolates separated them into three species with different habitat distributions, the widely distributed T. africanus and the more specialized, T. melanesiensis and T. affectus. The three Thermosipho species can also be differentiated on the basis of genome content. For instance the T. africanus genomes had the largest repertoire of carbohydrate metabolism, which could explain why these isolates were obtained from ecologically more divergent habitats. The three species also show different capacities for defense against foreign DNA. T. melanesiensis and T. africanus both had a complete RM system, while this was missing in T. affectus. These observations also correlated with Pacbio sequencing, which revealed a methylated T. melanesiensis BI431 genome, while no methylation was detected among two T. affectus isolates. All the genomes carry CRISPR arrays accompanied by more or less complete CRISPR-cas systems. Interestingly, some isolates of both T. melanesiensis and T. africanus carry integrated prophage elements, with spacers matching these in their CRISPR arrays. Taken together, the comparative genomic analyses of Thermosipho spp. revealed genetic variation allowing habitat differentiation within the genus as well as differentiation with respect to invading mobile DNA that is present in subsurface ecosystems.

microbiology↗