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

Ramunas Stepanauskas,Eric D Becraft,Arvydas Lubys,Maria G Pachiadaki,Jessica M Labonté,Tadas Povilaitis,Wendy K Bellows,Elizabeth A Fergusson,Nicole J Poulton,Brian P Thompson,Joseph Brown,Julia M Brown,Corianna J Mascena,Ben Tupper

doi:10.1038/s41467-017-00128-z

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.

Highlights

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms
Since its invention in 200225, multiple displacement amplification (MDA) has been the most widely used genomic DNA (gDNA) amplification method in Single-cell genomics (SCG) due to its multiple advantages: (a) long, overlapping amplicons that are well suited for genomic sequencing and subsequent de novo assembly; (b) high fidelity of the phi[29] polymerase; and (c) simple reaction setup that reduces the risk of handling errors and contamination and facilitates automation
Single-cell WGA-X reactions were significantly faster than MDA reactions containing the same templates, with an average critical point (Cp; the time required for reaching the inflection point of reaction’s exponential phase) of 1.9 vs. 9.6 h (Fig. 1b, c; p < 0.001, Student t-test)

Summary

Introduction

Microbial single-cell genomics can be used to provide insights into the metabolic potential, interactions, and evolution of uncultured microorganisms. 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. Several studies report reduced amplification biases through modified methods such as performing MDA in nano-liter-scale and pico-liter-scale liquid volumes[30,31,32] or in agarose gels[33], or employing protein priming[34] These approaches do not address the systemic MDA bias against high %GC templates and remain difficult to integrate into high-throughput workflows that involve sorting of specific cell types or single-cell phenotype analyses.

Methods

Results

Conclusion