Metabarcoding of the 16S rRNA gene is commonly used to characterize microbial communities, by estimating the relative abundance of microbes. Here, we present a method to retrieve the concentrations of the 16S rRNA gene per gram of any environmental sample using a synthetic standard in minuscule amounts (100 ppm to 1% of the 16S rRNA sequences) that is added to the sample before DNA extraction and quantified by two quantitative polymerase chain reaction (qPCR) reactions. This allows normalizing by the initial microbial density, taking into account the DNA recovery yield. We quantified the internal standard and the total load of 16S rRNA genes by qPCR. The qPCR for the latter uses the exact same primers as those used for Illumina sequencing of the V3‐V4 hypervariable regions of the 16S rRNA gene to increase accuracy. We are able to calculate the absolute concentration of the species per gram of sample, taking into account the DNA recovery yield. This is crucial for an accurate estimate as the yield varied between 40% and 84%. This method avoids sacrificing a high proportion of the sequencing effort to quantify the internal standard. If sacrificing a part of the sequencing effort to the internal standard is acceptable, we however recommend that the internal standard accounts for 30% of the environmental 16S rRNA genes to avoid the PCR bias associated with rare phylotypes. The method proposed here was tested on a feces sample but can be applied more broadly on any environmental sample. This method offers a real improvement of metabarcoding of microbial communities since it makes the method quantitative with limited efforts.

Dietary fiber content and composition affect microbial composition and activity in the gut, which in turn influence energetic contribution of fermentation products to the metabolic energy supply in pigs. This may affect feed efficiency (FE) in pigs. The present study investigated the relationship between the fecal microbial composition and FE in individual growing-finishing pigs. In addition, the effects of diet composition and sex on the fecal microbiome were studied. Fecal samples were collected of 154 grower-finisher pigs (3-way crossbreeds) the day before slaughter. Pigs were either fed a diet based on corn/soybean meal (CS) or a diet based on wheat/barley/by-products (WB). Fecal microbiome was characterized by 16S ribosomal DNA sequencing, clustered by operational taxonomic unit (OTU), and results were subjected to a discriminant approach combined with principal component analysis to discriminate diets, sexes, and FE extreme groups (10 high and 10 low FE pigs for each diet by sex-combination). Pigs on different diets and males vs. females had a very distinct fecal microbiome, needing only 2 OTU for diet (P = 0.020) and 18 OTU for sex (P = 0.040) to separate the groups. The 2 most important OTU for diet, and the most important OTU for sex, were taxonomically classified as the same bacterium. In pigs fed the CS diet, there was no significant association between FE and fecal microbiota composition based on OTU (P > 0.05), but in pigs fed the WB diet differences in FE were associated with 17 OTU in males (P = 0.018) and to 7 OTU in females (P = 0.010), with 3 OTU in common for both sexes. In conclusion, our results showed a diet and sex-dependent relationship between FE and the fecal microbial composition at slaughter weight in grower-finisher pigs.