Manzari, C. | Search bioRxiv

bioRxiv2022

Dealing with the promise of metabarcoding in mega-event biomonitoring: EXPO2015 unedited data

As human activities on our planet persist, causing widespread and irreversible environmental degradation, the need to biomonitor ecosystems has never been more pressing. These circumstances have required a renewal in monitoring techniques, encouraged by necessity to develop more rapid and accurate tools which will support timely observations of ecosystem structure and function. The World Exposition (from now EXPO2015) hosted in Milan from May to October 2015 was a global event that could be categorized as a mega-event, which can be defined as an acute environmental stressor, possibly generating biodiversity alteration and disturbance. During the six months of EXPO2015, exhibitors from more than 135 countries and 22 million visitors insisted on a 1.1 million square meters area. Faced with such a massive event, we explore the potential of DNA metabarcoding using three molecular markers to improve the understanding of anthropogenic impacts in the area, both considering air and water monitoring. Furthermore, we explore the effectiveness of the taxonomy assignment phase considering different taxonomic levels of analysis and the use of data mining approaches to predict sample origin. Unless the degree of taxa identification still remains open, our results showed that DNA metabarcoding is a powerful genomic-based tool to monitor biodiversity at the microscale, allowing us to capture exact fingerprints of specific event sites and to explore in a comprehensive manner the eukaryotic community alteration. With this work, we aim to disentangle and overcome the crucial issues related to the generalization of DNA metabarcoding in order to support future applications.

molecular biology↗

bioRxiv2021

Natural and after colon washing fecal samples: the two sides of the coin for investigating the human gut microbiome

To date there are several studies focusing on the importance of gut microbiome for human health, however the selection of a universal sampling matrix representative of the microbial biodiversity associated to the gastrointestinal (GI) tract, still represents a challenge. Here we present a study in which, through a deep metabarcoding analysis of the 16S rRNA gene, we compared two sampling matrices, feces (F) and colonic lavage liquid (LL), in order to evaluate their accuracy to represent the complexity of the human gut microbiome. A training set of 37 volunteers was attained and paired F and LL samples were collected from each subject. A preliminary absolute quantification of total 16S rDNA, performed by droplet digital PCR (ddPCR), confirmed that sequencing and taxonomic analysis were performed on same total bacterial abundance obtained from the two sampling methods. The taxonomic analysis of paired samples revealed that, although specific taxa were predominantly or exclusively observed in LL samples, as well as other taxa were detectable only or were predominant in stool, the microbiomes of the paired samples F and LL in the same subject hold overlapping taxonomic composition. Moreover, LL samples revealed a higher biodiversity than stool at all taxonomic ranks, as demonstrated by the Shannon Index and the Inverse Simpsons Index. We also found greater inter-individual variability than intra-individual variability in both sample matrices. Finally, functional differences were unveiled in the gut microbiome detected in the F and LL samples. A significant overrepresentation of 22 and 13 metabolic pathways, mainly occurring in Firmicutes and Proteobacteria, was observed in gut microbiota detected in feces and LL samples, respectively. This suggests that LL samples may allow for the detection of microbes adhering to the intestinal mucosal surface as members of the resident flora that are not easily detectable in stool, most likely representative of a diet-influenced transient microbiota. This first comparative study on feces and LL samples for the study of the human gut microbiome demonstrates that the use of both types of sample matrices may represent a possible choice to obtain a more complete view of the human gut microbiota in response to different biological and clinical questions.

molecular biology↗