Single Cell InsightsStudying environmental microbial communities cell by cell

Introduction

Dissecting complex microbial communities has incredible potential in the

quest to decipher the world around us and find new sources of enzymes,

antibiotics and other drugs. The challenge that remains is how to gain a

deeper understanding of the roles and interactions of individual microbes.

Advancements in whole genome amplification (WGA), next-generation

sequencing (NGS) and bioinformatics have enabled researchers to

delve into the sequence contents and metabolic functions of microbial

communities. These new metagenomic approaches have overcome the

limitations of microbial culture-dependent technologies.

What’s more, in the past few years, single cell sequencing has emerged

as an investigational approach with incredible potential. It provides a

view of the cell-by-cell community by separating out individual microbes

prior to sequencing. This enables in-depth analysis of the genetic

composition and potential of discrete organisms.

In this issue of Single Cell Insights, we provide short summaries of some

recent reviews and research papers focused on various applications

and methodologies for single cell sequencing of environmental microbial

communities.

Sample to Insight

Reviews

Clingenpeel, S., Clum, A., Schwientek, P., Rinke, C. and Woyke, T. (2014) Reconstructing each cell’s genome within complex

microbial communities – dream or reality? Front. Microbiol. 5, 771, doi: 10.3389/fmicb.2014.00771

This review summarizes the current state of single cell genomics technology for microbial communities, discussing its

potential for new applications.

Stepanauskas, R. (2015) Wiretapping into microbial interactions by singe cell genomics. Front. Microbiol. 6, 258, doi:

10.3389/fmicb.2015.00258

The author gives his opinions on the importance of single cell genomics as applied to diverse microbial studies.

Research papers

Mansor, M., Hamilton, T.L., Fantle, M.S. and Macalady, J.L. (2015),

Metabolic diversity and ecological niches of Achromatium populations

revealed with single-cell genomic sequencing. Front. Microbiol., 6, 822,

doi: 10.3389/fmicb.2015.00822

Single cell sequencing was applied to reveal the anabolic and

metabolic potential of the bacterial genus Achromatium and the genetic

heterogeneity of Achromatium populations. Cells were enriched from

sediment samples of a spring, flow-sorted, and handpicked using a

micromanipulator. WGA of individual cells was performed with the

REPLI-g® Single Cell Kit. WGA DNA samples containing 16S rRNA gene

sequences affiliated with the genus Achromatium were then analyzed via

whole genome sequencing. Draft genomes with 80% completeness were

achieved. The draft genomes are a basis for additional analyses of the

metabolic versatility of Achromatium and the study yielded new insights

for further discussions on the mechanisms of intracellular calcification.

Start with just one cell

A typical bacterial cell contains only a few

femtograms of genomic DNA, which is far

less than typically required as sample input

for NGS and other analytical methods. A

WGA step offers accurate amplification

with highly uniform genome coverage

for improved genome assembly and high

confidence in sequence accuracy.

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Chen, Y.R. et al. (2016) Novel species and expanded distribution of ellipsoidal multicellular magnetotactic prokaryotes.

Environ. Microbiol. Rep. 8(2), 218–226

This study focused on a specific morphotype of multicellular magnetotactic prokaryotes (MMPs), which are groups of 10

to 100 cells of diverse magnetotactic bacteria. Phylogenetic analyses of these MMPs were performed. First, the specific

morphotype (ellipsoidal MMPs) was isolated via micromanipulation, followed by WGA using the REPLI-g Single Cell Kit and

16S rRNA gene-based sequence analysis. The phylogenetic analysis revealed similarities and differences in the species

composition of MMPs from various sampling sites, indicating a new candidate species.

Eloe-Fadrosh, E.A. et al. (2016) Global metagenomic survey reveals a

new bacterial candidate phylum in geothermal springs. Nature Comm.

7, 10476, doi: 10.1038/ncomms10476

The researchers discovered a novel bacterial candidate phylum:

Candidatus Kryptonia using metagenomic data mining and single cell

genomics data from samples collected from geothermal springs. The

single cell sampling was performed from four geothermal springs.

Single cells from the samples were isolated using FACS sorting, followed

by WGA with the REPLI-g Single Cell Kit, PCR screening for genomes

from single cells matching Canditatus Kryptonia SSU rRNA sequences,

and sequencing on an Illumina MiSeq® instrument. Eighteen single

cell genomes with an average genome completeness of 67% were

recovered. The high-quality draft genomes from the metagenomic data

analysis and single cell genomics enabled further analyses of bacteria–

virus interactions and the detection of a novel fusion between two

different CRISPR–Cas types.

Optimized for single bacterial cells

The chemistry of the REPLI-g Single

Cell Kit is optimized to ensure superior

performance, even with a single bacterial

cell. Its multiple displacement amplification

(MDA) technology has proven useful in

a whole range of published studies of

individual environmental microbe cells.

The buffers and reagents undergo a

controlled decontamination procedure

ensuring the elimination of detectable

residual DNA contamination, making

it especially suited for such sensitive

applications.

Studying environmental microbial communities cell by cell 06/2016 3

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at www.qiagen.com or can be requested from QIAGEN Technical Services or your local distributor.

Trademarks: QIAGEN®, Sample to Insight®, REPLI-g® (QIAGEN Group); Illumina®, MiSeq® (Illumina, Inc.).

1103321 06/2016 PROM-9831-001 © 2016 QIAGEN, all rights reserved.

Want to learn more about single cell sequencing solutions from QIAGEN? Visit www.qiagen.com/SingleCellAnalysis.

Nakamura, K. et al. (2016) Culture-independent method for identification of microbial enzyme encoding genes by activity-

based single-cell sequencing using a water-in-oil microdroplet platform, Scientific Reports 6, 22259 doi: 10.1038/srep22259

An activity-based single cell sequencing approach enabled the visual evaluation of microbial cells during screening and offered

a new, targeted approach for specific isolation of microbial cells. Environmental microbes were encapsulated in water–oil

microdroplets containing a fluorogenic substrate for a target enzyme. This enabled screening for microdroplets containing cells

that were active for the specific enzyme. The selected microbial cells were recovered and subjected to WGA using the REPLI-g

Single Cell Kit. Successfully amplified single cell genomes were further analyzed via whole genome sequencing.

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