Single-Cell sequencing: limitations,
applications and technical advances
Alex Subias Gusils
Subject: Genomics
Master in Advanced Genetics
7/01/2015
Introduction
Single-cell sequencing
- Consist on analyzing the whole sequence information from
an individual cell using NGS
- The NGS techniques can help to improve it
Using the sc-seq we can:
Sequence whole or partial genomes or transcriptomes from single cells
Study of the diversity of cells at the individual cell level
Obtain a higher resolution of cellular differences
Introduction
Single-cell sequencing is a relatively recent technique:
Major developments and landmarks in human genetics and genomics
Naidoo N et al., 2001
The sc-seq needs time to achieve the objectives proposed at the beginning
Methods
There are two different methods:
Single-cell Genome Sequencing (scDNA-seq)
Single-cell RNA Sequencing (scRNA-seq)
The scDNA-seq protocol has five essential steps:
Cell isolation
DNA extraction
DNA amplification
Micromanipulation
Fluorescent-activated
cell sorting (FACS)
or
Laser-capture
microdissection (LCM)
Analysis
Sequencing
PAPER 2
Methods
There are two different methods:
Single-cell Genome Sequencing (scDNA-seq)
Single-cell RNA Sequencing (scRNA-seq)
- It provides the expression profile of individual cells analyzing their transcriptomes
- Identify rare cell types within a cell population
Analysis
The scheme of the protocol is the following:
Cell isolation
RNA extraction
cDNA synthesis
cDNA amplification
Sequencing
Considerations
We have some limitations in two of the steps of the protocols:
Isolation of single cells
-There are different methods to obtain isolate cells and each one
has its advantages and disadvantages
- Laser-capture microdissection (LCM)
It is used to identify the cells of interest in an stained
section. We can cut it with a UV laser and then transfer
onto a membrane
Is hard to capture a whole single cell without also collecting
the materials from neighbouring cells
Saliba AE et al., 2014
Considerations
- Micromanipulation using a patch pipette
It is useful to isolate cells from culture or liquid
samples such as saliva or blood
It is laborious and time-consuming
Navin N et al., 2011
- Fluorescent-activated cell sorting (FACS)
The most commonly used method. We use fluorescent
labeled antibodies to isolate cells of interest according
to the targeted cell-surface markers
We need antibodies that target specific proteins and
that’s not always possible to obtain
Saliba AE et al., 2014
Considerations
Genome or cDNA amplification
- Genome and transcriptome sequencing require more starting material than what is found
in an individual cell so heavy amplification is often needed
- This huge amplification could result in degradation, sample loss and contamination and it
can have an effect on sequence quality and robustness
DNA
Amplified DNA
Applications
Studies of heterogeneous samples
- We can identify different cell types in scarce samples in disease like cancer
Identify differences between healthy and diseased tissues
Cell identity
Navin N et al., 2011
- The single-cell transcriptome profiling can identify biologically relevant differences in cells,
even when cells may not be distinguishable by cell morphology
Microbial genetics
- The data obtained from microorganisms might establish processes for
culturing in the future
Applications
Cancer prognosis
- The number of CTCs in peripheral blood of cancer patients has been shown to correlate to prognosis
- Using the scRNA-seq we can differentiate CTCs from normal blood cells
Study of somatic mutations
-We can discover and screen somatic mutations that play an important
role in the origin and progression of diseases such as aging, immunity,
cancer, neurodegenerative disorders and others
Navin N et al., 2011
Disease evolution
- The scDNA-seq can reveal mutations and structural changes in the genome of cancer cells
- This information can be used to describe their clonal structure and to trace the evolution and
spread of the disease (metastasis)
Future
- LESS AMPLIFICATION
- MORE CELLS
- MORE TYPE OF DATA
Conclusions
 The single-cell sequencing is a new technique that needs time to obtain important
results
 There has been considerable recent progress in analyzing single-cell genomes and
mRNA transcriptomes
 Improvements in the basic technology as well as in data analysis and interpretation
will be important for obtaining the precision of measurement needed to understand
the role of a cell
References
 Eberwine J, Sul J-Y, Bartfai T, Kim J (2014). ‘The promise of single-cell sequencing’, Nature Methods
 Lovett M (2013). ‘The applications of single-cell genomics’. Human Molecular Genetics
 Macaulay IC, Voet T (2014). ‘Single cell genomics: advances and future perspectives’. PLoS Genetics
 Naidoo N, Pawitan Y, Soong R, Cooper DN, Ku CS (2011). ‘Human genetics and genomics a decade after
the release of the draft sequence of the human genome’. Human Genomics
 Navin N, Hicks J (2011) . ‘Future medical applications of singles-cell sequencing in cancer’.
Genome Medicine
 Navin N, Kendall J, Troge J, Andrews P et al. (2011). ‘Tumour evolution inferred by
single-cell sequencing’, Nature
 Nawy T. (2013). ‘Single-cell sequencing’. Nature Methods
 Saliba AE, Westermann AJ, Gorski SA, Vogel J (2014). ‘Single-cell RNA-seq: advances and future challenges’.
Nucleic Acids Research