16S rDNA sequencing of BALB/c and C57BL/7 mouse gut microbiomes
ABSTRACT: Age-dependent changes of the gut-associated microbiome have been linked to increased frailty and systemic inflammation. This study found that age-associated changes of the gut microbiome of BALB/c and C57BL/6 mice could be reverted by co-housing of aged (22 months old) and adult (3 months old) mice for 30-40 days or faecal microbiota transplantation (FMT) from adult into aged mice. This was demonstrated using high-throughput sequencing of the V3-V4 hypervariable region of bacterial 16S rRNA gene isolated from faecal pellets collected from 3-4 months old adult and 22-23 months old aged mice before and after co-housing or FMT.
Project description:The evolutional trajectory of gut microbial colonization from birth has been shown to prime for health later in life. Here, we combined cultivation-independent 16S rRNA gene sequencing and metaproteomics to investigate the functional maturation of gut microbiota in faecal samples from full-term healthy infants collected at 6 and 18 months of age. Phylogenetic analysis of the metaproteomes showed that Bifidobacterium provided the highest number of distinct protein groups. Considerable divergences between taxa abundance and protein phylogeny were observed at all taxonomic ranks. Age had a profound effect on early microbiota where compositional and functional complexity of less dissimilar communities increased with time. Comparisons of the relative abundances of proteins revealed the transition of taxon-associated saccharolytic and carbon metabolism strategies from catabolic pathways of milk and mucin-derived monosaccharides feeding acetate/propanoate synthesis to complex food sugars fuelling butyrate production. Furthermore, co-occurrence network analysis uncovered two anti-correlated modules of functional taxa. A low-connected Bifidobacteriaceae-centred guild of facultative anaerobes was succeeded by a rich club of obligate anaerobes densely interconnected around Lachnospiraceae, underpinning their pivotal roles in microbial ecosystem assemblies. Our findings establish a framework to visualize whole microbial community metabolism and ecosystem succession dynamics, proposing opportunities for microbiota-targeted health-promoting strategies early in life.
Project description:A phylogenetic microarray targeting 66 families described in the human gut microbiota has been developped aud used to monitor the gut microbiota's structure and diversity. The microarray format provided by Agilent and used in this study is 8x15K. A study with a total of 4 chips was realized. Arrays 1 and 2: Hybridization with 100ng of labelled 16S rRNA gene amplicons from a mock community sample and 250ng of labelled 16S rRNA gene amplicons from 1 faecal sample. Each Agilent-030618 array probe (4441) was synthetized in three replicates. Arrays 3 and 4: Hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 faecal samples. Each Agilent-40558 array probe (4441) was synthetized in three replicates.
Project description:Comparison of faecal flora of three healthy individuals and a patient suffering from Ulcerative Colitis during disease and remission states. Faecal samples were taken and frozen at -80 within one hour.
Project description:Multiomics of faecal samples collected from individuals in families with multiple cases of type 1 diabetes mellitus (T1DM) over 3 or 4 months. Metagenomic and metatranscriptomic sequencing and metaproteomics were carried out, as well as whole human genome sequencing. Phenotypic data is available.
Project description:Microbiota from rats fed with wheat aleurone and plant omega fatty acids In this study we investigated how an AX-rich WA and ALA from linseed oil (LO) modulate the gut microbiota of rats. Wistar rats were fed a standard diet and received either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Feacal samples were recovered after the 12 week treatments. DNA extractions were performed using using the Qiagen's DNA Stool Kit (Qiagen, West Sussex, UK). 10ng of DNA template were amplified by PCR (16S gene) and purified using Qiagen's Qiaquick PCR purification kit (Qiagen, West Sussex, UK). 1ug of purified PCR product were labelled with either Cy3 or Cy5 using Genomic DNA ULS Labelling kit (Agilent Technologies, Palo Alto, CA). 250ng of labelled DNA were hybridized on the microarray for 24h at 65°C. Washings were performed as recommended by the manufacturer. Microarray scanning was performed on a Surescan Microarray scanner (Agilent Technologies, Palo Alto, CA). Data were extracted using the Feature extraction software (Agilent Technologies, Palo Alto, CA). The retained intensity value for each probe was the ratio between the spot’s median intensity signals and the median of background signals. A 13 chip study was realized to analyze the feacal microbiota of rats treated with either an iso-energetic control oil (PO), control oil + aleurone (A+PO), linseed oil (LO) or linseed oil + aleurone (A+LO) during 12 weeks. Each microarray corresponding to hybridization with 250ng of labelled 16S rRNA gene amplicons from 2 rat DNA faecal samples. Microbiota structure and diversity were assessed using the HuGChip (Tottey et al., 2013). Each probe (4441) was synthetized in three replicates. On the same array, 2 different samples were hybridized. One labelled with the Cy3 dye and one with the Cy5 dye. The results were processed as single channel (13 raw data files available on Series records for 25 samples).
Project description:We compared the microbiota of paired mouse caecal contents and faeces by applying a multi-omic approach, including 16S rDNA sequencing, shotgun metagenomics, and shotgun metaproteomics. The aim of the study was to verify whether faecal samples are a reliable proxy for the mouse colonic luminal microbiota, as well as to identify changes in taxonomy and functional activity between caecal and faecal microbial communities, which have to be carefully considered when using stool as sample for mouse gut microbiota investigations.
Project description:A three-stage continuous fermentative system was developed to simulate and control physicochemical factors of the gut biology. Inoculation was of each reactor was performed from a human fecal sample which was initially amplified with a batch procedure. Samples from the initial feces, the batch and from the bioreactors media were collected to extract bacterial DNA. 16S PCR amplification was performed to assess the microbial diversity at the family level using the HuGChip. Amplified DNA was purified and labelled with either Cy3 or Cy5 dye and hybridized on the microarray. A 5 chip study was realized, each corresponding to hybridization with 250ng of labelled 16S rRNA gene amplicons from either the initial stool, the batch inoculum or fermentative medium different compartments of the simulated colon (Proximal, Transversal and Distal). Each probe (4441) was synthetized in three replicates.
Project description:This study in rats was designed to investigate whether whole rhye (WR) can influence the metabolism of n-3 and n-6 long-chain fatty acids (LCFA) and gut microbiota composition. For 12 weeks, rats were fed a diet containing either 50% WR or 50% refined rye (RR). Total bacterial DNA was extracted from fecal and cecal samples (n=5 per group). 16S PCR amplification was performed to assess the microbial diversity at the family level using the HuGChip. Amplified DNA was purified and labelled with either Cy3 or Cy5 dye and hybridized on the microarray. A 15 chip study was realized, each corresponding to hybridization with 250ng of labelled 16S rRNA gene amplicons from either mice fecal and cecal samples. Each probe (4441) was synthetized in three replicates.
Project description:Satellite cells are responsible for the long-term regenerative capacity of adult skeletal muscle. The diminished muscle performance and regenerative capacity of aged muscle is thought to reflect progressive fibrosis and atrophy. Whether this reduction in muscle competency also involves a diminishment in the intrinsic regulation of satellite cell self-renewal remains unknown. We used microarray to identify gene expression changes underlying the marked reduction in the capacity of satellite cells to self-renew, contribute to regeneration and repopulate the niche as they age. Skeletal muscles from heterozygous Pax7-ZsGreen mice were isolated at defined stages: E17.5 (fetal - whole forelimb and hindlimb), postnatal day 21 (adolescent - hindlimb), 2-3 month old (young adult - hindlimb) and >1 year old (older adult - hindlimb) mice. ZsGreen-positive skeletal muscle satellite cells were isolated by FACS and pooled (fetal n=4, adolescent n=6, young adult n=8 and older adult n=8 mice).
Project description:Transcriptional profiling of the microdissected SVZ from 7-month-old mice Adult neurogenesis is suppressed in the SVZ of 3xTg mice, a model of Alzheimer's disease. To better understand the underlying mechanisms of this suppression, the goals of this experiment were to compare the transcriptional profiles of the SVZ neural stem cell niche in 3xTg-AD mice versus strain controls. We used early middle-aged mice (7-months-old) rather than old mice, in order to identify genetic changes that are not caused secondarily to other degenerative changes occurring in these mice. Two-condition experiment, 3xTg vs WT SVZ. Biological replicates: 4 for each.