Combining Flow Cytometry and Metagenomics Improves Recovery of Metagenome-Assembled Genomes in a Cell Culture from Activated Sludge

Abstract

The recovery of metagenome-assembled genomes is biased towards the most abundant species in a given community. To improve the identification of species, even if only dominant species are recovered, we investigated the integration of flow cytometry cell sorting with bioinformatics tools to recover metagenome-assembled genomes. We used a cell culture of a wastewater microbial community as our model system. Cells were separated based on fluorescence signals via flow cytometry cell sorting into sub-communities: dominant gates, low abundant gates, and outer gates into subsets of the original community. Metagenome sequencing was performed for all groups. The unsorted community was used as control. We recovered a total of 24 metagenome-assembled genomes (MAGs) representing 11 species-level genome operational taxonomic units (gOTUs). In addition, 57 ribosomal operational taxonomic units (rOTUs) affiliated with 29 taxa at species level were reconstructed from metagenomic libraries. Our approach suggests a two-fold increase in the resolution when comparing sorted and unsorted communities. Our results also indicate that species abundance is one determinant of genome recovery from metagenomes as we can recover taxa in the sorted libraries that are not present in the unsorted community. In conclusion, a combination of cell sorting and metagenomics allows the recovery of MAGs undetected without cell sorting.

Genome-centric analyses of 165 metagenomes show that mobile genetic elements are crucial for the transmission of antimicrobial resistance genes to pathogens in activated sludge and wastewater

“…Another study by Feng et al ( 49 ) retrieved MAGs from the chicken gut microbiome from public repositories and predicted ARGs without comparing the diversity of MAGs recovered from each sample and location. In addition, previous studies determined the factors affecting genome recovery in which low sequencing depth was reported as a critical feature in MAGs recovery ( 18 , 50 ).…”

“…The development of high-throughput sequencing technologies has made it possible to sequence the entire DNA content of microbial communities, a process known as metagenomics ( 18 – 20 ). Metagenomics has drastically expanded our knowledge of uncultivable microorganisms and their functional potential ( 18 , 21 , 22 ).…”

“…The development of high-throughput sequencing technologies has made it possible to sequence the entire DNA content of microbial communities, a process known as metagenomics ( 18 – 20 ). Metagenomics has drastically expanded our knowledge of uncultivable microorganisms and their functional potential ( 18 , 21 , 22 ). Additionally, this approach improved the taxonomic classification of microbial communities up to the species level while allowing the discovery of novel functional genes in the microbiome ( 22 , 23 ).…”

Nanosensor Chemical Cytometry: Advances and Opportunities in Cellular Therapy and Precision Medicine

“…FSC primarily reflects the size or volume of the cells, while SSC provides insight into the internal structure of the cells including granularity. 25,26 Many researchers have used flow cytometry to analyze the therapeutic characteristics of various cell types such as leukocytes and MSCs, based on fluorescence associated with specific labels. 27−29 These tools allow for the discrimination of different cell populations as well as measurement of cell viability and activation status by assessing the fluorescence intensity of single cells.…”

“…The cell fluid is surrounded by detectors, one aligned with the laser beam for measuring forward scatter (FSC), and the other positioned at a right angle to the stream for measuring side scatter (SSC). FSC primarily reflects the size or volume of the cells, while SSC provides insight into the internal structure of the cells including granularity. , Many researchers have used flow cytometry to analyze the therapeutic characteristics of various cell types such as leukocytes and MSCs, based on fluorescence associated with specific labels. − These tools allow for the discrimination of different cell populations as well as measurement of cell viability and activation status by assessing the fluorescence intensity of single cells. With its ability to provide detailed cellular analysis, flow cytometry plays a crucial role in advancing the study of cell therapy.…”