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One of the ongoing controversies is how to study the bacterial composition in complex ecosystems.To date, some of the widely used approaches to characterize the intestinal microbiota are: metagenomics, phylogenetic microarrays, DNA fingerprinting techniques, and q PCR .This study describes the design, optimization, and validation of the Gut Microbiotassay: a primer setup consisting of 24 primer sets targeting the main bacterial phyla of the intestinal microbiota at various taxonomic levels, to be used with the AA48.48 in combination with NGS.Furthermore, it demonstrates the applicability of the Gut Microbiotassay on luminal content collected from the small and large intestine of piglets of different diarrhoeic status.
Afterwards, amplicons can be harvested directly from the AA48.48 sample inlets where the respective samples were initially loaded.
454-sequencing confirmed the specificity of the primer sets.
Diarrhoea was associated with a reduced number of members from the genus The Gut Microbiotassay provides fast and affordable high-throughput quantification of the bacterial composition in many samples and enables further descriptive taxonomic information if combined with 454-sequencing.
Especially metagenomic approaches are receiving increased attention in the study of microbial communities as a result of their shorter sequencing speed, extended read length, and lower costs .
The Access Array 48.48, AA48.48, (Fluidigm Corporation, South San Francisco, CA, USA) creates an affordable link between high-throughput q PCR and next generation sequencing (NGS) and provides manageable data with valuable quantitative and taxonomic information.
An interplate calibrator (IPC) was included in all AA48.48, consisting of bacterial DNA extracted from ~ 100 mg colonic content from a healthy conventional pig, 14-week-old, Danish landrace.