Midi-metagenomics: A novel approach for cultivation independent microbial genome reconstruction from environmental samples

Abstract

As the majority of environmental microbial organisms still evade cultivation attempts, genomic insights into many taxa are limited to cultivation-independent approaches. However, current methods of metagenomic binning and single cell genome sequencing have individual drawbacks, which can limit the quality as well as completeness of the reconstructed genomes. Current attempts to combine both approaches still use whole genome amplification techniques which are known to be prone to bias. Here we propose a novel approach for the purpose of metagenomic genome reconstructions, that utilizes the potential of fluorescence-activated cell sorting (FACS) for targeted enrichment and depletion of different cell types to create distinct cell fractions of sufficient size circumvent amplification. By distributing sequencing efforts over these fractions as well as the original sample, co-assemblies become highly optimized for co-abundance variation based binning approaches. 'Midi-metagenomics' enables accurate metagenome assembled genome (MAG) reconstruction from individual sorted samples with higher quality than co-assembly of multiple distinct samples and has potential for the targeted enrichment and sequencing of uncultivated organism of interest.

Targeted Cell Labeling and Sorting of Prokaryotes for Cultivation and Omics Approaches

“…Fluorescence-Activated Cell Sorting FACS technology relies on the detection of fluorescence signals caused by the cells themselves (autofluorescence), by fluorescently labeled antibodies or ligands that bind to certain cell-associated molecules. Besides fluorescence, simple scattering of light can also be detected and used for characterization of cells in terms of size and complexity/granularity as detected by forward scatter (FSC) and side scatter (SSC) [Veal et al, 2000;Luo et al, 2020;Vollmers et al, 2023]. Using a droplet deflection technique, FACS sorters are capable of sorting tens of thousands of cells per second enabling high throughput analyses to detect, count as well as sort subfractions of a complex heterogenous sample [Veal et al, 2000] (Fig.…”

“…In this approach, cells are fractionated into larger pools of several million cells in regard to different properties which can be distinguished by flow cytometers (e.g., different shape and size, metabolic, or physiological markers). From these pools, DNA can be directly isolated and sequenced resulting in assemblies that are suitable for co-variance-based binning approaches, yielding MAGs of significantly higher quality compared to conventional metagenomic concepts and avoiding additional costs for amplification [Vollmers et al, 2023].…”