37
are tedious when handling microbial samples from various sources (Rinke et al.
2014; Gawad et al. 2016). Automated micromanipulation approaches consisting of
micromechanical valves in microfluidic devices are coming to the forefront of
application under single-cell genomics (White et al. 2011; Leung et al. 2012;
Macosko et al. 2015; Gawad et al. 2016).
Whatever the method applied, it is essential to check that an individual cell has
been isolated properly to avoid a false interpretation. In an ideal case, this can be
achieved through microscopic data of each single cell per well. Furthermore, identification of scalable approaches for separating single cells demands active research
that may lead to the development of precise tools for all capture performance metrics (Gawad et al. 2016).
4.1.2 Whole-Genome Amplification
For obtaining significant genetic information from single cells, amplification of a
single copy of the genome along with reducing different artifacts such as amplification biases, genome loss, mutations, and chimeras are essentially required. In
one approach, the complete genome from targeted single cells is equipped with
PCR- based amplification by using a universal sequence throughout the genome
(Lichter et al. 1990), a universal sequence ligated to sheared genome (Troutt et al.
1992), or degenerate or random oligonucleotide priming (Telenius et al. 1992;
Zhang et al. 1992). The second strategy involves methods based on isothermal
approaches and multiple-displacement amplification (Zhang et al. 2001).
Furthermore, to remove the poor resolution of PCR approaches and isothermal
biases, new methods involve restricted isothermal amplification then subjected to
PCR of previously amplified products (Zong et al. 2012). These combined
approaches are at the forefront of the WGA methods in existing single-cell studies
(Gawad et al. 2016).
4.1.3 Interrogation of WGA Products
Application of a single technique under single-cell study is not sufficient to produce
whole-genome amplicons and copy number estimation. The kind of genomic interrogation opted for the study of single-cell genomics is chosen after considering the
deviation of whole-genome amplification (Gawad et al. 2016; De Bourcy et al.
2014). Study of a location-specific genome within a single cell may assist us to find
the physiologically active area that confers concise and coherent results and to save
the cost and time of adopted techniques. Small and fewer variant genomes have
minimum chances of including the technical deviations and errors that generally
occur during the initial cycles of complete genome amplification. These technical
deviations lead to incorrect results that often produce erroneous positive signals
4.1 Single-Cell Genomics (SCG)
are tedious when handling microbial samples from various sources (Rinke et al.
2014; Gawad et al. 2016). Automated micromanipulation approaches consisting of
micromechanical valves in microfluidic devices are coming to the forefront of
application under single-cell genomics (White et al. 2011; Leung et al. 2012;
Macosko et al. 2015; Gawad et al. 2016).
Whatever the method applied, it is essential to check that an individual cell has
been isolated properly to avoid a false interpretation. In an ideal case, this can be
achieved through microscopic data of each single cell per well. Furthermore, identification of scalable approaches for separating single cells demands active research
that may lead to the development of precise tools for all capture performance metrics (Gawad et al. 2016).
4.1.2 Whole-Genome Amplification
For obtaining significant genetic information from single cells, amplification of a
single copy of the genome along with reducing different artifacts such as amplification biases, genome loss, mutations, and chimeras are essentially required. In
one approach, the complete genome from targeted single cells is equipped with
PCR- based amplification by using a universal sequence throughout the genome
(Lichter et al. 1990), a universal sequence ligated to sheared genome (Troutt et al.
1992), or degenerate or random oligonucleotide priming (Telenius et al. 1992;
Zhang et al. 1992). The second strategy involves methods based on isothermal
approaches and multiple-displacement amplification (Zhang et al. 2001).
Furthermore, to remove the poor resolution of PCR approaches and isothermal
biases, new methods involve restricted isothermal amplification then subjected to
PCR of previously amplified products (Zong et al. 2012). These combined
approaches are at the forefront of the WGA methods in existing single-cell studies
(Gawad et al. 2016).
4.1.3 Interrogation of WGA Products
Application of a single technique under single-cell study is not sufficient to produce
whole-genome amplicons and copy number estimation. The kind of genomic interrogation opted for the study of single-cell genomics is chosen after considering the
deviation of whole-genome amplification (Gawad et al. 2016; De Bourcy et al.
2014). Study of a location-specific genome within a single cell may assist us to find
the physiologically active area that confers concise and coherent results and to save
the cost and time of adopted techniques. Small and fewer variant genomes have
minimum chances of including the technical deviations and errors that generally
occur during the initial cycles of complete genome amplification. These technical
deviations lead to incorrect results that often produce erroneous positive signals
4.1 Single-Cell Genomics (SCG)