The generation of the largest to date pig gut metagenomic dataset and the insights gained into microbiome composition and function

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Early weaning and intensive farming practices predispose piglets to the development of infectious and often lethal diseases, against which antibiotics are used. Besides contributing to the build-up of antimicrobial resistance, antibiotics are known to substantially alter the gut microbial composition. While extensive literature reports on the dysbiotic effects of orally administered antibiotics, fewer studies established the effects of intramuscular antibiotic treatment on the gut community, and none, to our knowledge, studied the effects of intramuscular neomycin. In order to study the effects of intramuscular neomycin, and of two probiotic formulations, we sampled 911 faecal microbial communities from 126 post-weaning piglets for the duration of 5 weeks. The processing of such a large amount of samples required the optimization of costs with the maintenance of sample quality. To this end, we developed a sequencing library preparation technique, called Hackflex, which allows the generation of Illumina-compatible sequencing libraries with up to an 11-fold cost reduction. The sequencing generated the largest piglet gut microbiome dataset up to date, comprising of 8Tb of metagenomic shotgun sequence data, corresponding to 27 billion read pairs. A phylogenetic framework allowed us to interrogate the data. We found factors such as breed, litter and tiny differences in age to be associated with differences in the microbiome and we determined the persistence of those correlations. Specific microbial compositional changes were found to correlate with the probiotic and the antibiotic treatments. Lastly, reads were pooled and co-assembled by host, and the differential coverage abundance information was used in the process of binning, to produce metagenomic assembled genomes (MAGs). This method constructs draft genomes without the need for reference databases, which are unequivocally biased towards better-represented organisms. Clustered MAGs allowed us to track microbial shifts at the species- and strain-level occurring between the 3rd and the 9th week of life of post-weaning piglets, and the functional analysis on the carbohydrate enzyme repertoire substantially expanded the current knowledge of species-to-enzyme mapping. The contribution of this work to the current knowledge of the post-weaning gut microbial development opens the doors to the possibility to optimise the chances of survival of specific strains by knowing exactly when they should be administered; and to predict a strain’s substrate utilization and in such way, engineer probiotic and prebiotic cocktails for the improvement of substrate energy efficiency. These possibilities are applicable to the human (e.g. diet) as well as the livestock settings (e.g. diet, methane emission control).
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