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