MiSeq-derived artificial sequences appeared to be of good quality, thus bioinformatics tools failed to remove MiSeq artefacts. Even after removing singleton sequences or operational taxonomic units (OTUs), it is not clear how many sequence artefacts remained. Here, 16S rRNA genes were amplified from soil, human feces, pig feces, and groundwater. These were sequenced with five separate runs of MiSeq. Subsequently, each run of MiSeq was compared through alpha and beta-diversity analyses. We found more than half the OTUs were not in consensus through the multiple MiSeq runs, resulting in varying group-specific biomarker OTUs in each MiSeq run. Thus, differential abundance test should be interpreted with caution, and we suggest that results also should be verified further with other quantification methods such as qPCR.
In this study, we investigated the effects of fasting on gut microbiota of mice fed normal (CTL) or high-fat diets (HF). Mice were raised for 16 weeks and fasted for a day at the end of the experiment. Fecal samples were collected one day before and after fasting, which were analyzed using MiSeq. Our results showed that the species richness and evenness were decreased in fasted HF group, whereas no difference was observed for CTL groups. Moreover, HF fed mice gut microbiota showed different microbial communities after fasting, while CTL groups did not show microbiota shifts. Differential abundance analysis showed that fasting CTL group mice increased and decreased one operational taxonomic unit (OTU) in S24_7 and one OTU in Ruminococcaceae, respectively. On the other hand, fasting HF group mice decreased 10 OTUs and increased 3 OTUs most of which were classified to Ruminococcaceae. Our results suggest that fasting mice may affect the abundance of Ruminococcaceae species and effects of fasting seem to be more obvious for HF-fed mice compared to those of mice fed CTL-diet.
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