Biology subjects

Ahn-Jarvis, J.

Publications and source records attributed to Ahn-Jarvis, J..

5 recordsLinked to original sources

bioRxiv2026

Enriching the Human Stool Microeukaryotes for Shotgun Sequencing

The human gut microbiome harbours a diverse community of microeukaryotes, predominantly fungi, which may potentially play important roles in gut ecology and homeostasis. Despite their potential, the study of gut microeukaryotes has been hampered by the limited sensitivity of standard sequencing approaches, which struggle to capture DNA from low-abundance microorganisms against the overwhelming background of bacterial biomass. To address this, we developed a method to selectively enrich for microeukaryotic cells in human faecal samples by depleting bacterial cells prior to metagenomic sequencing. Through systematic comparison and optimisation at each processing step, we established a robust standard operating procedure (SOP) for microeukaryotic cell enrichment. By benchmarking this SOP across eight human faecal samples with three technical replicates each, we showed that it consistently increased microeukaryote representation in metagenomic libraries, greater microeukaryotic taxonomic diversity, and a reduced proportion of unclassified taxa. Together, these improvements enabled substantially deeper characterisation of the microeukaryotic fraction of the human gut microbiome.

microbiology↗

bioRxiv2025

Optimizing storage, high-molecular weight DNA extraction and genome reconstructions from human faecal samples.

Metagenomic sequencing has become an essential tool for assessing the composition and functional potential of microbial communities. However, established metagenomic DNA extraction protocols are limited by trade-offs between universal taxonomic representation, DNA quality, quantity and molecule length, making them mostly unsuitable for 3rd generation sequencing. MethodsWe assessed the effectiveness of high molecular weight (HMW) DNA workflows from faecal samples, using different combinations of collection, storage and extraction kits. Faecal samples were either immediately processed, frozen at -80{degrees}C or stored in DNA/RNA Shield, GutAlive or OMNIgeneGUT and then subjected to three different DNA isolation kits, each modified for mechanical, chemical and enzymatic cell lysis steps, totalling 32 evaluated combinations. Isolated DNA was assessed for its quality and quantity, while taxonomic consistency was evaluated using metataxonomics and validated with metagenomic sequencing. ResultsThe yield of HMW DNA differed substantially between storage and DNA extraction protocols. Specifically, storage protocol had the highest impact on HMW DNA recovery, with DNA/RNA Shield storage yielding on average 51% more HMW DNA >30kb. Similarly, the sample storage had independently the effect size of R2 = 26.9% on the microbiome composition, and since the DNA extraction protocol in our study inherently combines both DNA extraction kit and lysis type, the latter had the largest effect size (R2 = 12.8%). While all kits had specific dis/advantages and biases, DNA/RNA Shield and Maxwell RSC PureFood GMO and Authentication kit performed best overall for long-read metagenomics. The protocol was validated on a) isolate genomes and b) 4 faecal samples, demonstrating both increased DNA yield at >30kb molecule length. Using the MG-TK pipeline these metagenomes yielded genomes on average, per sample, 48 Pacific Biosciences (PacBio) and 55 Oxford Nanopore Technologies (ONT) circular bacterial metagenomic assembled genomes. ConclusionThe current gold-standard of freezing faecal samples was surprisingly inconsistent and inefficient for long-read metagenomics, necessitating rethinking gut microbial study designs. We provide a simple and cheap protocol that preserves both taxonomic composition and HMW DNA quality, while our bioinformatics workflow allows for complete bacterial genome reconstruction.

molecular biology↗

bioRxiv2024

Modelling the gut microbiota of children with malnutrition: in vitro models reveal differences in fermentability of widely consumed carbohydrates

There is increasing evidence in children suffering from Severe Acute Malnutrition (SAM) that there is disruption of the gut microbiome and low gut microbiota diversity, which may be contributing factors to poor outcomes during nutritional treatment and recovery. The gut microbiome of children with SAM has been demonstrated to have a lower production of beneficial short chain fatty acids, which may contribute to impaired gut barrier function. Recently, several microbiota-directed therapies have been tested in clinical trials in children with SAM. Among them we hypothesized that feeds containing fermentable carbohydrates from various sources (legumes, chicory, milk oligosaccharides) would be fermented to produce beneficial microbial metabolites by the microbiota of children with SAM. In this study we used an in vitro model system inoculated with stool from children with SAM to investigate the fermentability of four substrates; inulin (a chicory-derived fructan), two milk powders (one supplemented with a human milk oligosaccharide) and a chickpea enriched feed. We demonstrated that while the milk powders and chickpea feed were fermented to produce short chain fatty acids, inulin was only fermented to a very limited degree. Through 16S rRNA sequencing we demonstrated that the samples inoculated with inulin had low microbial diversity and linked this to the limited ability to metabolise inulin. Through revealing the fermentability of different complementary feeds, the findings of this study will be of use for the design of future therapeutic feeds for treatment of SAM. ImportanceMalnutrition is a major contributor to childhood mortality globally and is a major public health problem primarily affecting Lower- and Middle-Income Countries. Despite the development of nutritional recovery therapies, for those with the severe and complicated form of malnutrition (SAM), mortality and relapse rates remain high. Emerging evidence suggests a role for the gut microbiome in these poor outcomes, which is known to be significantly altered in children in SAM, compared to healthy age matched controls. To aid in recovery from SAM, nutritional interventions should be designed to support the gut microbiome, using a range of ingredients targeted for colonic fermentation. It is important to understand the fermentation capacity of the gut microbiome of children with SAM, to design future nutritional interventions. In this work, we demonstrate that inulin, a widely used chicory-derived prebiotic, is not a suitable fermentation substrate for the gut microbiome of SAM children, while legume-based formulations and milk oligosaccharides result in increased production of beneficial metabolites.

microbiology↗

bioRxiv2021

Linking carbohydrate structure with function in the human gut microbiome using hybrid metagenome assemblies

BackgroundComplex carbohydrates that escape digestion in the small intestine, are broken down in the large intestine by enzymes encoded by the gut microbiome. This is a symbiotic relationship between particular microbes and the host, resulting in metabolic products that influence host gut health and are exploited by other microbes. However, the role of carbohydrate structure in directing microbiota community composition and the succession of carbohydrate-degrading microbes is not fully understood. Here we take the approach of combining data from long and short read sequencing allowing recovery of large numbers of high quality genomes, from which we can predict carbohydrate degrading functions, and impact of carbohydrate on microbial communities. ResultsIn this study we evaluate species-level compositional variation within a single microbiome in response to six structurally distinct carbohydrates in a controlled model gut using hybrid metagenome assemblies. We identified 509 high-quality metagenome-assembled genomes (MAGs) belonging to ten bacterial classes and 28 bacterial families. We found dynamic variations in the microbiome amongst carbohydrate treatments, and over time. Using these data, the MAGs were characterised as primary (0h to 6h) and secondary degraders (12h to 24h). Annotating the MAGs with the Carbohydrate Active Enzyme (CAZyme) database we are able to identify species which are enriched through time and have the potential to actively degrade carbohydrate substrates. ConclusionsRecent advances in sequencing technology allowed us to identify significant unexplored diversity amongst starch degrading species in the human gut microbiota including CAZyme profiles and complete MAGs. We have identified changes in microbial community composition in response to structurally distinct carbohydrate substrates, which can be directly related to the CAZyme complement of the enriched MAGs. Through this approach, we have identified a number of species which have not previously been implicated in starch degradation, but which have the potential to play an important role.

microbiology↗

bioRxiv2021

STARCH SYNTHASE 4 is required for normal starch granule initiation in amyloplasts of wheat endosperm

O_LIStarch granule initiation is poorly understood at the molecular level. The glucosyltransferase, STARCH SYNTHASE 4 (SS4), plays a central role in granule initiation in Arabidopsis leaves, but its function in cereal endosperms is unknown. We investigated the role of SS4 in wheat, which has a distinct spatiotemporal pattern of granule initiation during grain development. C_LIO_LIWe generated TILLING mutants in tetraploid wheat (Triticum turgidum) that are defective in both SS4 homoeologs. The morphology of endosperm starch was examined in developing and mature grains. C_LIO_LISS4 deficiency led to severe alterations in endosperm starch granule morphology. During early grain development, while the wild type initiated single A-type granules per amyloplast, most amyloplasts in the mutant formed compound granules due to multiple initiations. This phenotype was similar to mutants deficient in B-GRANULE CONTENT 1 (BGC1). SS4 deficiency also reduced starch content in leaves and pollen grains. C_LIO_LIWe propose that SS4 and BGC1 are required for the proper control of granule initiation during early grain development that leads to a single A-type granule per amyloplast. The absence of either protein results in a variable number of initiations per amyloplast and compound granule formation. C_LI

plant biology↗