Abstract
Optofluidic single-cell genome amplification was used to obtain genome sequences from sub-micron cells collected from the euphotic and mesopelagic zones of the northwestern Sargasso Sea. Plankton cells were visually selected and manually sorted with an optical trap, yielding 20 partial genome sequences representing seven bacterial phyla. Two organisms, E01-9C-26 (Gammaproteobacteria), represented by four single cell genomes, and Opi.OSU.00C, an uncharacterized Verrucomicrobia, were the first of their types retrieved by single cell genome sequencing and were studied in detail. Metagenomic data showed that E01-9C-26 is found throughout the dark ocean, while Opi.OSU.00C was observed to bloom transiently in the nutrient-depleted euphotic zone of the late spring and early summer. The E01-9C-26 genomes had an estimated size of 4.76–5.05 Mbps, and contained “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes. Metabolic reconstruction indicated E01-9C-26 are likely versatile methylotrophs capable of scavenging C1 compounds, methylated compounds, reduced sulfur compounds, and a wide range of amines, including D-amino acids. The genome sequences identified E01-9C-26 as a source of “O” and “W”-type monooxygenase genes related to methane and ammonium monooxygenases that were previously reported from ocean metagenomes, but are of unknown function. In contrast, Opi.OSU.00C genomes encode genes for catabolizing carbohydrate compounds normally associated with eukaryotic phytoplankton. This exploration of optofluidics showed that it was effective for retrieving diverse single-cell bacterioplankton genomes and has potential advantages in microbiology applications that require working with small sample volumes or targeting cells by their morphology.
Highlights
Single-cell genomics (SCG) and metagenomics are well-proven and effective sources of insight into microbial processes in nature
In this study we explored the application of optofluidics to marine plankton, testing refinements to our optical trapping design and optimized multiple-displacement amplification (MDA) reaction conditions (Landry et al, 2013)
The findings show that optofluidics was effective in this application, and may have future uses in microbiology when small sample size and cell visualization are important factors
Summary
Single-cell genomics (SCG) and metagenomics are well-proven and effective sources of insight into microbial processes in nature. Single cell genome amplification by optofluidics is a relatively underutilized technology that uses manual sorting with an optical trap to retrieve genomes from low volume cell suspensions. Cells or small cell aggregates that have been injected into a microfluidic chip are selected by the operator on the basis of morphology or other characteristics visible through a microscope, and moved to reaction chambers within the chip, where the genome amplification is carried out. Aspects of optofluidics that can be an advantage in some applications include very small sample and reaction volumes and visual cell selection (Marshall et al, 2012). Despite potential advantages in some applications, single cell genome amplification by optofluidics has remained relatively rare because fluorescence-activated cell sorting (FACS) approaches using flow cytometry have been very successful and have a much higher throughput rate
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