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
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
- The NGS techniques can help to improve it
Using the sc-seq we can:
Naidoo N et al., 2001
Introduction
Single-cell sequencing is a relatively recent technique:
Major developments and landmarks in human genetics and genomics
The sc-seq needs time to achieve the objectives proposed at the beginning
Methods
Single-cell Genome Sequencing (scDNA-seq)
There are two different methods:
Single-cell RNA Sequencing (scRNA-seq)
The scDNA-seq protocol has five essential steps:
Laser-capture microdissection (LCM)
Cell isolation
Micromanipulation
Fluorescent-activated cell sorting (FACS)
DNA extraction
or
DNA amplification
SequencingAnalysis
PAPER 2
Methods
Single-cell Genome Sequencing (scDNA-seq)
There are two different methods:
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
The scheme of the protocol is the following:
Cell isolation RNA extraction cDNA synthesis cDNA amplification Sequencing
Analysis
Considerations
Saliba AE et al., 2014
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
Considerations
Saliba AE et al., 2014
Navin N et al., 2011
- Micromanipulation using a patch pipette
- Fluorescent-activated cell sorting (FACS)
It is useful to isolate cells from culture or liquid
samples such as saliva or blood
It is laborious and time-consuming
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
Considerations
DNA Amplified DNA
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
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
- The single-cell transcriptome profiling can identify biologically relevant differences in cells,
even when cells may not be distinguishable by cell morphology
Navin N et al., 2011
Microbial genetics
- The data obtained from microorganisms might establish processes for
culturing in the future
Navin N et al., 2011
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
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
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
There has been considerable recent progress in analyzing single-cell genomes and
mRNA transcriptomes
The single-cell sequencing is a new technique that needs time to obtain
important
results
References
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
Eberwine J, Sul J-Y, Bartfai T, Kim J (2014). ‘The promise of single-cell sequencing’, Nature Methods
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
Saliba AE, Westermann AJ, Gorski SA, Vogel J (2014). ‘Single-cell RNA-seq: advances and future challenges’.
Nucleic Acids Research
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