Biology subjects

Eisenhofer, R.

Publications and source records attributed to Eisenhofer, R..

9 recordsLinked to original sources

bioRxiv2026

Impact of sequencing approach on ecological inference from highly variable bat gut microbiomes

Understanding the functional potential of animal gut microbiomes requires analytical approaches that can accurately capture both taxonomic and functional diversity, particularly in hosts with samples with low and variable microbial DNA fractions, such as bats. Here, we compared 16S amplicon sequencing and genome-resolved metagenomics for characterising the gut microbiomes of three sympatric insectivorous bats (Pipistrellus kuhlii, Hypsugo ariel, and Cnephaeus bottae). Amplicon sequencing recovered 3,536 amplicon sequence variants (ASVs) spanning 29 microbial phyla, including five archaeal groups, while metagenomics yielded 135 metagenome-assembled genomes (MAGs) across 15 bacterial phyla. Alpha and beta diversity patterns differed significantly between approaches. Applying prevalence- and abundance-based filtering to amplicon data improved alpha diversity congruence with metagenomic profiles, indicating that low-abundance and rare ASVs inflate diversity estimates, while the low microbial DNA fractions in bat faeces challenge genome reconstruction of such taxa. Functional reconstructions diverged notably, with metagenomics-based functional profiles yielding consistently lower metabolic capacities than those indirectly inferred from 16S amplicon sequencing, both before and after prevalence- and abundance-based filtering, due to incompleteness of many reconstructed genomes. However, metagenome-assembled genomes harboured metabolic capacities missed by the indirect functional inference of metabarcoding, which largely rely on genomes of strains characterised in model organisms. Together, these findings highlight the influence of chosen methodology, host species, and data filtering on microbiome profiling in taxa with low microbial biomass. We propose practical guidelines to improve analytical consistency and reliability when studying gut microbiomes of bats and other hosts with similarly variable microbial communities.

microbiology↗

bioRxiv2024

Functional insights into the effect of feralisation on the gut microbiota of cats worldwide

Feralisation, the process by which domesticated organisms revert to a wild state, is a widespread phenomenon across various species. Successfully adapting to a new environment with different access to food, shelter, and other resources requires rapid physiological and behavioural changes, which could potentially be facilitated by gut microbiota plasticity. To investigate whether alterations in gut microbiota support this transition to a feral lifestyle, we analysed the gut microbiomes of domestic and feral cats from six geographically diverse locations using genome-resolved metagenomics. By reconstructing 229 draft genomes from 92 cats, we identified a typical carnivore microbiome structure, with notable diversity and taxonomic differences across regions. While overall diversity metrics did not differ significantly between domestic and feral cats, hierarchical modelling of species communities, accounting for geographic and sex covariates, revealed distinct taxonomic and functional profiles between the two groups. While taxonomic enrichment was balanced, microbial functional capacities were significantly enriched in feral cats. These functional enhancements, particularly in amino acid and lipid degradation, correspond to feral cats dietary reliance on crude protein and fat. Additionally, functional differences were consistent with behavioural contrasts, such as the more aggressive and elusive behaviour measured in feral cats compared to the docile behaviour of domestic cats. Finally, the observed enrichment in short-chain fatty acid, neurotransmitter, and vitamin B12 production in feral cats aligns with improved cognitive function and potentially contributes to their heightened aggression and elusiveness. Our findings suggest that microbiome shifts may play a significant role in the development of physiological and behavioural traits advantageous for a feral lifestyle, supporting the adaptive success of feral cats in the wild.

evolutionary biology↗

bioRxiv2024

Blanking on Blanks: Few Insect Microbiota Studies Control for Contaminants - A Systematic Review

Research on insect-microbe relationships is booming, with DNA sequencing being the most commonly used method to describe insect microbiota. However, sequencing is vulnerable to contamination, especially when the sample has low microbial biomass. Such low-biomass samples are common across insect taxa, developmental stages, and tissue types. Identifying putative contaminants is essential to distinguish between true microbiota and introduced contaminant DNA. It is therefore important that studies control for contamination, but how often this is done is unknown. To investigate the status quo of contamination control, we undertook a systematic literature review to quantify the prevalence of negative control usage and contamination control across the literature on insect microbiota (specifically bacterial communities) over the past 20 years. Two-thirds of the 243 insect microbiota studies evaluated did not include blanks (negative controls), and only 13.6% of the studies sequenced these blanks to control for contamination in their samples. Our findings highlight a major lack of contamination control in the field of insect microbiota research. This result suggests that a large number of microbes reported in the literature may be contaminants as opposed to insect-associated microbiota, and that more rigorous contamination control is needed to improve research reliability, validity and reproducibility. Based on our findings, we recommend a modified version of the guidelines outlined in the RIDES checklist: Report methodology, Include negative controls, Determine the level of contamination, Explore contamination downstream, and State the amount of off-target amplification. IMPORTANCEOur systematic review reveals a major lack of methodological rigour within the field of research on insect-associated microbiota. The small percentage of studies that control for contamination suggests that a considerable proportion of bacteria reported in the literature could be contaminants. The implication of this finding is that true microbiota may be masked or misrepresented, especially in insects with low microbial biomass.

microbiology↗

bioRxiv2024

Quantifying microbial DNA in metagenomes improves microbial trait estimation

Shotgun metagenomics is a powerful tool for studying the genomic traits of microbial community members, such as genome size, gene content, etc. While such traits can be used to better understand the ecology and evolution of microbial communities, the accuracy of their estimations can be critically influenced by both known and unknown factors. One factor that can bias trait estimations is the proportion of eukaryotic DNA in a metagenome, as some bioinformatic tools assume that all DNA reads in a metagenome are non-eukaryotic. Here, we help resolve a recent debate about the influence of eukaryotic DNA in the estimation of average genome size from a global soil sample dataset using a new bioinformatic tool. Contrary to what was assumed, our reanalysis of this dataset revealed that soil samples can contain a substantial proportion of eukaryotic DNA ([~]38.8%), which severely inflated average genome size estimates. We report that correcting for this bias significantly improves the statistical support for the negative relationship between average bacterial genome size and soil pH. These results highlight that metagenomes can contain large quantities of eukaryotic DNA, and that new methods that correct for this can improve microbial trait estimation.

microbiology↗

bioRxiv2024

Large-scale estimation of bacterial and archaeal DNA prevalence in metagenomes reveals biome-specific patterns

Metagenomes often contain many reads derived from eukaryotes. However, there is usually no reliable method for estimating the prevalence of non-microbial reads in a metagenome, forcing many analysis techniques to make the often-faulty assumption that all reads are microbial. For instance, the success of metagenome-assembled genome (MAG) recovery efforts is assessed by the number of reads mapped to recovered MAGs, a procedure which will underestimate the true fidelity if eukaryotic reads are present. Here we present "SingleM microbial_fraction" (SMF), a scalable algorithm that robustly estimates the number of bacterial and archaeal reads in a metagenome, and the average microbial genome size. SMF does not use eukaryotic reference genome data and can be applied to any Illumina metagenome. Based on SMF, we propose the "Domain-Adjusted Mapping Rate" (DAMR) as an improved metric to assess microbial genome recovery from metagenomes. We benchmark SMF on simulated and real data, and demonstrate how DAMRs can guide genome recovery. Applying SMF to 136,284 publicly available metagenomes, we report substantial variation in microbial fractions and biome-specific patterns of microbial abundance, providing insights into how microorganisms and eukaryotes are distributed across Earth. Finally, we show that substantial amounts of human host DNA sequence data have been deposited in public metagenome repositories, possibly counter to ethical directives that mandate screening of these reads prior to release. As the adoption of metagenomic sequencing continues to grow, we foresee SMF being a valuable tool for the appraisal of genome recovery efforts, and the recovery of global patterns of microorganism distribution.

bioinformatics↗

bioRxiv2024

Pouch microbiome changes during lactation in the short-beaked echidna (Tachyglossus aculeatus)

Monotreme and marsupial early development is characterised by a short gestation, the birth of the young at an early stage of development, and a long lactation in the pouch or pseudo-pouch. The lack of a functional adaptive immune system in these altricial young raises questions about how they survive in a microbe-rich world. Previous studies on marsupial pouches have revealed changes to pouch microbe composition during lactation but no information is available in monotremes. We investigated changes in the echidna pseudo-pouch microbiome during different stages of the reproductive cycle and whether this differs between wild and zoo-managed animals. Swab samples were obtained from wild and captive echidna pseudo-pouches outside of breeding season, during courtship and breeding, and during lactation. 16S rRNA gene metabarcoding revealed that the pseudo-pouch microbiome undergoes dramatic changes during lactation, with a reduction in bacterial taxa that may be pathogenic. These changes were not observed in samples taken outside of breeding season or during courtship and mating. This showed that the echidna pseudo-pouch environment changes during lactation to accommodate young that lack a functional adaptive immune system. This study pioneers pouch microbiome research in monotremes, provides new biological information on echidna reproduction, and may also provide information about the effects of captive management to inform breeding programs in the future.

genomics↗

bioRxiv2023

A comparison of short-read, HiFi long-read, and hybrid strategies for genome-resolved metagenomics

Shotgun metagenomics enables the reconstruction of complex microbial communities at a high level of detail. Such an approach can be conducted using both short-read and long-read sequencing data, as well as a combination of both. To assess the pros and cons of these different approaches, we used 22 faecal DNA extracts collected weekly for 11 weeks from two respective lab mice to study seven performance metrics over four combinations of sequencing depth and technology: i) 20 Gbp of Illumina short-read data, ii) 40 Gbp of short-read data, iii) 20 Gbp of PacBio HiFi long-read data, and iv) 40 Gbp of hybrid (20 Gbp of short-read + 20 Gbp of long-read) data. No strategy was best for all metrics, but instead, each one excelled across different metrics. The long-read approach yielded the best assembly statistics, with the highest N50 and lowest number of contigs. The 40 Gbp short-read approach yielded the highest number of refined bins. Finally, the hybrid approach yielded the longest assemblies, and the highest mapping rate to the bacterial genomes. Our results suggest that while long-read sequencing significantly improves the quality of reconstructed bacterial genomes, it is more expensive and requires deeper sequencing than short-read approaches to recover a comparable amount of reconstructed genomes. The most optimal strategy is study-specific, and depends on how researchers assess the tradeoff between the quantity and quality of recovered genomes. ImportanceOur understanding of microbial communities is limited by the technologies we employ. Here, we test several different DNA sequencing techniques to better understand the pros and cons of each. Long read DNA sequencing allowed for the reconstruction of higher quality and even complete microbial genomes, however, the cost was greater than commonly used short-read DNA sequencing. We suggest researchers consider the trade-offs between each method and decide based on the goals of their research question/s.

microbiology↗

bioRxiv2022

Neanderthals did not likely consume Physcomitrium patens - a model moss species

This manuscript is discussing the method of Weyrich et al., 2017, "Neanderthal behaviour, diet, and disease inferred from ancient DNA in dental calculus". When studying the dietary profile of a Neanderthal specimen from El Sidron cave (Spain) by sequencing ancient DNA present in calcified dental plaque (calculus) the authors identified a wide-range of potential food sources, including woolly rhinoceros, mushrooms, pine nuts, and moss - namely the less-than-abundant model species Physcomitrium patens. We doubted that Neanderthals were actually eating P. patens. By analyzing the ancient DNA reads using different mapping methods, we show likely a misinterpretation based on the previously used methods. The probability of Neanderthals eating P. patens is the same as eating rice or tomato. However, neither crop was grown in Europe at the time Neanderthals thrived.

evolutionary biology↗

bioRxiv2018

Laboratory contamination over time during low-biomass sample analysis

Bacteria are not only ubiquitous on earth but can also be incredibly diverse within clean laboratories and reagents. The presence of both living and dead bacteria in laboratory environments and reagents is especially problematic when examining samples with low endogenous content (e.g. skin swabs, tissue biopsies, ice, water, degraded forensic samples, or ancient material), where contaminants can outnumber endogenous microorganisms within samples. The contribution of contaminants within high-throughput studies remains poorly understood because of the relatively low number of contaminant surveys. Here, we examined 144 negative control samples (extraction blank and no-template amplification controls) collected in both typical molecular laboratories and an ultraclean ancient DNA laboratory over five years to characterize long-term contaminant diversity. We additionally compared the contaminant content within a homemade silica-based extraction method, commonly used to analyse low-endogenous samples, with a widely used commercial DNA extraction kit. The contaminant taxonomic profile of the ultraclean ancient DNA laboratory was unique compared to the modern molecular biology laboratories, and changed over time according to researchers, month, and season. The commercial kit contained higher microbial diversity and several human-associated taxa in comparison to the homemade silica extraction protocol. We recommend a minimum of two strategies to reduce the impacts of laboratory contaminants within low-biomass metagenomic studies: 1) extraction blank controls should be included and sequenced with every batch of extractions and 2) the contributions of laboratory contamination should be assessed and reported in each high-throughput metagenomic study.

microbiology↗