Improved genome recovery and integrated cell-size analyses of individual uncultured microbial cells and viral particles

Abstract

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. Here we present WGA-X, a method based on multiple displacement amplification of DNA that utilizes a thermostable mutant of the phi29 polymerase. WGA-X enhances genome recovery from individual microbial cells and viral particles while maintaining ease of use and scalability. The greatest improvements are observed when amplifying high G+C content templates, such as those belonging to the predominant bacteria in agricultural soils. By integrating WGA-X with calibrated index-cell sorting and high-throughput genomic sequencing, we are able to analyze genomic sequences and cell sizes of hundreds of individual, uncultured bacteria, archaea, protists, and viral particles, obtained directly from marine and soil samples, in a single experiment. This approach may find diverse applications in microbiology and in biomedical and forensic studies of humans and other multicellular organisms.

Interfacial Nanoinjection‐Based Nanoliter Single‐Cell Analysis

“…As shown in Figure , the sizes of the assemblies ranged from 0.21 to 3.67 Mbp, and the genome completeness was estimated to vary from 6.04% to 73.5% (Figure S8a, Supporting Information). Compared to previous single‐cell MDA methods, the FACS‐INJ pipeline produces comparable assembly size and completeness with markedly lower contamination levels (<5%), indicating that our single‐cell WGA is robust and compatible with environmental samples. We also found large numbers of carbohydrate‐active enzymes (CAZymes) in all single‐cell genome assemblies (Figure S8b, Supporting Information), indicating that these microbes potentially function in carbon cycling in deep oceans.…”

“…Recent research advances have promoted the study of biological systems with respect to their physiological phenotypes, genomics, transcriptomics, epigenomics, proteomics, multiomics, and biodiversity at the single‐cell level. Single‐cell analysis allows the fundamental understanding of cell‐to‐cell heterogeneity and provides unprecedented resolution in complex biological systems, especially those from rare cell types or uncultivated microbes . However, current approaches for single‐cell analysis usually involve complicated processes and instruments, require the use of microfabrication devices, and are cost‐prohibitive for common laboratories.…”

Single cell genomes of Prochlorococcus, Synechococcus, and sympatric microbes from diverse marine environments

“…This workflow was evaluated for assembly errors using a series of benchmark cultures with diverse genome complexity and %GC 21 .…”

Assessing the viral content of uncultured picoeukaryotes in the global‐ocean by single cell genomics

“…However, the application of these approaches to characterize virus‐host interactions will miss intraspecific genetic variability of both actors. More recently, several new MDA‐like methods, such as WGA‐X (Stepanauskas et al, ), TruePrime (Picher et al, ) or REPLI‐g (Ahsanuddin et al, ) have been developed for improving the genome recovery from single environmental cells (bacterial, archaeal and protists) and viral particles with high GC‐content genomes. Compared with the conventional MDA, these amplification alternatives may provide a better genome recovery of microbial taxa, including some not amenable to standard MDA (Stepanauskas et al, ).…”