Part C | 20.2
512 Part C Marine Genomics
into separate chambers, where each sorted cell was
lysed individually. The genome of each individual cell
was then amplified by MDA in 50 nL volumes [20.29].
Amplified genomic DNA from each cell was then
separately recovered from the chip and sequenced
by high-throughput DNA pyrosequencing. Combining SCG genome amplification with metagenomics,
Blainey et al. sequenced and identified the genome of
Candidatus Nitrosoarchaeum limnia SFB1, an AOA
from low-salinity sediments in San Francisco Bay,
using single-cell and metagenomic genome sequence
data [20.39]. Another interesting example demonstrates
the possibility of combining different genome analysis techniques in the same chip for marine cell genome
analysis. Leung et al. [20.42] developed a microfluidic
device capable of running multiple single-cell applications, such as phenotypic sorting of bacteria followed
by clonal analysis of growth rates, taxonomic identification of single bacteria by small subunit ribosomal
ribonucleic acid (RNA) gene quantitative PCR (qPCR)
and sequencing, and high-throughput single-cell whole
genome amplification (WGA) and sequencing. Interestingly, this device, shown in Fig. 20.2c, combines
a programmable droplet-based microfluidic device with
the reconfigurable flow-routing capabilities of integrated microvalve technology. It is composed of 95
individual microchambers, a reagent-metering module, a cell-sorting module, and an integrated nozzle
that allows for automated recovery of on-chip reaction products without cross-contamination. A threevalve peristaltic pump is used to deliver arbitrary
picoliter droplets of eight separated reagents in discrete increments from eight separate inlets. Reagent
droplets are dispensed directly into a flowing stream
of carrier fluid; delivered to a selected storage chamber it merges with any previously dispensed droplets.
When a cell of interest is identified, it is pumped
into a droplet and delivered to a microchamber where
it can be analyzed by qPCR and WGA. This device
was used to analyze the genome of single cells and
microbial consortia from diverse environmental samples, such as marine enrichment cultures and deep-sea
sediments.
Cell Analysis
A variety of analytical methods have been deployed to
identify and study populations of marine microorganisms with a view to understanding both the organisms
and their behavior. Optical microscopy is generally
used to study marine microorganisms but the large ratio between the size of the target cell and the size of
Fig. 20.2a–d Microfluidic system for genome, cell, and
whole organism studies. (a) Top: DNA purification chip
and schematic diagram of one instance of the DNA isolation process. Bottom: multiplex digital PCR platform for
single-cell analysis in the environmental sample for amplification of all bacteria 16S rDNA and termite cluster 16S
rDNA (red fluorescence) and clone H FTHFS gene (green
fluorescence), (after [20.9] and [20.38]). (b) Left: picture
of the single-cell genome amplification device. Right: stepby-step process (after [20.35]). (c) Programmable dropletbased microfluidic device applied to multiparameter analysis of single marine microbes and microbial communities.
Left: schematic representation of the entire chip. Right
(from top to bottom): storage element geometry for droplet
immobilization and coalescence by flow-controlled wetting. Micrograph of a water droplet in a single chamber.
Graphic representation of the cell-sorting module and of
the reagent-metering module (after [20.42]). (d) Microfluidic chips for fish embryology study. Top: schematic representation of microfluidic segment technique for screening
and development studies on fish embryos. Bottom: photo
and 3-D streamline of flow obtained by computational fluid
dynamic simulations across the fully loaded device. Timelapse images of developing fish embryos collected every
24 h (after [20.43, 44]) I
the culture system (plate, petri dish, or tube) makes
the study labor intensive and time consuming. In the
early 1980s, flow cytometry was introduced as a complementary technique to analyze mixed microbial populations [20.30, 31, 45]. However, the high cost and the
large size of the apparatus limited its use as a standard tool for laboratory-based research and in situ
analysis. Toward the realization of a portable and affordable microfluidic cytometer, Benazzi et al. [20.46]
have developed a microfluidic chip capable of highspeed analysis of phytoplankton. The chip was made
from a glass substrate with planar microelectrodes patterned on the surface. A microfluidic channel made of
polydimetylsiloxane (PDMS) was bonded on the surface of the patterned glass substrate, perpendicularly
to the microelectrodes. The chip was placed above
a microscope objective and the cells were pumped
into the microchannel with the use of a syringe pump.
The phytoplankton flowed through the detection zone
where optical and electrical properties were measured
simultaneously. When a mixture of different species
of phytoplankton (here Synechococcus sp., Rhodosorus
m., and I. galbana) were introduced into the chip, measurement of the optical properties differentiated cells
in function of their autofluorescence, while electrical
512 Part C Marine Genomics
into separate chambers, where each sorted cell was
lysed individually. The genome of each individual cell
was then amplified by MDA in 50 nL volumes [20.29].
Amplified genomic DNA from each cell was then
separately recovered from the chip and sequenced
by high-throughput DNA pyrosequencing. Combining SCG genome amplification with metagenomics,
Blainey et al. sequenced and identified the genome of
Candidatus Nitrosoarchaeum limnia SFB1, an AOA
from low-salinity sediments in San Francisco Bay,
using single-cell and metagenomic genome sequence
data [20.39]. Another interesting example demonstrates
the possibility of combining different genome analysis techniques in the same chip for marine cell genome
analysis. Leung et al. [20.42] developed a microfluidic
device capable of running multiple single-cell applications, such as phenotypic sorting of bacteria followed
by clonal analysis of growth rates, taxonomic identification of single bacteria by small subunit ribosomal
ribonucleic acid (RNA) gene quantitative PCR (qPCR)
and sequencing, and high-throughput single-cell whole
genome amplification (WGA) and sequencing. Interestingly, this device, shown in Fig. 20.2c, combines
a programmable droplet-based microfluidic device with
the reconfigurable flow-routing capabilities of integrated microvalve technology. It is composed of 95
individual microchambers, a reagent-metering module, a cell-sorting module, and an integrated nozzle
that allows for automated recovery of on-chip reaction products without cross-contamination. A threevalve peristaltic pump is used to deliver arbitrary
picoliter droplets of eight separated reagents in discrete increments from eight separate inlets. Reagent
droplets are dispensed directly into a flowing stream
of carrier fluid; delivered to a selected storage chamber it merges with any previously dispensed droplets.
When a cell of interest is identified, it is pumped
into a droplet and delivered to a microchamber where
it can be analyzed by qPCR and WGA. This device
was used to analyze the genome of single cells and
microbial consortia from diverse environmental samples, such as marine enrichment cultures and deep-sea
sediments.
Cell Analysis
A variety of analytical methods have been deployed to
identify and study populations of marine microorganisms with a view to understanding both the organisms
and their behavior. Optical microscopy is generally
used to study marine microorganisms but the large ratio between the size of the target cell and the size of
Fig. 20.2a–d Microfluidic system for genome, cell, and
whole organism studies. (a) Top: DNA purification chip
and schematic diagram of one instance of the DNA isolation process. Bottom: multiplex digital PCR platform for
single-cell analysis in the environmental sample for amplification of all bacteria 16S rDNA and termite cluster 16S
rDNA (red fluorescence) and clone H FTHFS gene (green
fluorescence), (after [20.9] and [20.38]). (b) Left: picture
of the single-cell genome amplification device. Right: stepby-step process (after [20.35]). (c) Programmable dropletbased microfluidic device applied to multiparameter analysis of single marine microbes and microbial communities.
Left: schematic representation of the entire chip. Right
(from top to bottom): storage element geometry for droplet
immobilization and coalescence by flow-controlled wetting. Micrograph of a water droplet in a single chamber.
Graphic representation of the cell-sorting module and of
the reagent-metering module (after [20.42]). (d) Microfluidic chips for fish embryology study. Top: schematic representation of microfluidic segment technique for screening
and development studies on fish embryos. Bottom: photo
and 3-D streamline of flow obtained by computational fluid
dynamic simulations across the fully loaded device. Timelapse images of developing fish embryos collected every
24 h (after [20.43, 44]) I
the culture system (plate, petri dish, or tube) makes
the study labor intensive and time consuming. In the
early 1980s, flow cytometry was introduced as a complementary technique to analyze mixed microbial populations [20.30, 31, 45]. However, the high cost and the
large size of the apparatus limited its use as a standard tool for laboratory-based research and in situ
analysis. Toward the realization of a portable and affordable microfluidic cytometer, Benazzi et al. [20.46]
have developed a microfluidic chip capable of highspeed analysis of phytoplankton. The chip was made
from a glass substrate with planar microelectrodes patterned on the surface. A microfluidic channel made of
polydimetylsiloxane (PDMS) was bonded on the surface of the patterned glass substrate, perpendicularly
to the microelectrodes. The chip was placed above
a microscope objective and the cells were pumped
into the microchannel with the use of a syringe pump.
The phytoplankton flowed through the detection zone
where optical and electrical properties were measured
simultaneously. When a mixture of different species
of phytoplankton (here Synechococcus sp., Rhodosorus
m., and I. galbana) were introduced into the chip, measurement of the optical properties differentiated cells
in function of their autofluorescence, while electrical