Drilling down to the single-cell level provides insights into complex biological systems and diseases. Learn about single-cell sequencing, its applications and challenges.
Single-cell sequencing: an overview
Standard bulk methods of cell analysis use many thousands of cells. When we analyze that data, we’re effectively averaging out any small cell-to-cell variances and concentrating on features or data points that rise above the noise.
It’s easy to assume that all cells of the same timepoint from the same sample are, well, the same. But that’s far from the case. Cell populations are heterogeneous: studying single-cell samples is crucial to understanding these complex biological systems. Drilling down to the single-cell level allows us to understand the effects on and contributions by individual cells within their environment.
What is single-cell genomics?
Single-cell genomics applies standard analytical techniques, including sequencing and microarrays, to the individual cell, utilizing advanced techniques for selecting and handing individual cells and maximizing the raw material (DNA, RNA, proteins) held within. Single-cell genomics has numerous applications in both basic research and clinical settings.
Single-cell genomics: applications
- The heterogeneity of solid tumors is well-known. Single-cell sequencing gives researchers the ability to study individual cells from various points in a tumor’s progression and its microenvironment, and opens up investigative pathways that may lead to better diagnostics, treatments and cures.
- In cases where a direct biopsy would be invasive, single-cell sequencing enables clinicians to detect and monitor circulating tumor cells (CTCs), which present cancer biomarkers that can direct treatment, minimizing therapies that are unlikely to succeed.
- Liquid biopsies such as in non-invasive pre-natal testing (NIPT), are also quite well-established for sequencing cell-free DNA. In this example of liquid biopsy, NIPT can detect various genetic conditions from fetal DNA that circulates in the mother’s bloodstream, avoiding invasive testing.
- Single-cell genomics can be an essential tool in forensic applications where a few cells might be all you have to work with from a casework sample.
As the technology improves and becomes more accessible, the areas of application will only expand. Single-cell genomics is already allowing archaeologists, anthropologists and paleontologists utilize genomics in new and interesting ways.
Single-cell genomics: challenges
Three core processes in single-cell sequencing present challenges which can also affect sequencing outcomes.
- Selecting and handling individual cells
- Extracting DNA (ro\\or cDNA) from the cells
- Amplifying the genetic material.
Careful handling through manual pipetting might be the way to go if you’re working with a few individual cells. However, If you need to analyze a large sample at the single-cell level, this approach is quite labor intensive. In that case, microfluidics might be the best option. Recent developments enable these systems to handle thousands of cells in parallel.
Current extraction techniques are quite robust, though they do need careful control for efficient release and high yield of material for amplification.
Amplification is the most challenging process in single-cell sequencing. While there are several methods available, each technique can introduce bias that may affect your results.
Single-cell sequencing: methods of amplifying DNA
There are currently three general approaches to amplifying DNA:
- PCR-based amplification (Polymerase chain reaction-based amplification)
- Multiple displacement amplification (MDA)
- Combinations of PCR and MDA, such as MALBAC
All these methods have pros and cons, but it’s possible to manage the limitations with an understanding of how they work, and where the limitations come from.
PCR uses thermal cycling to induce DNA replication. Unfortunately, it is prone to variations in reliability across different loci, and false positives and negatives in analysis. This method has fallen out of use.
MDA amplifies DNA through multiple displacement, binding primers to newly-formed DNA while polymerization is still ongoing. MDA with generic primers and DNA polymerase Phi29 can amplify picograms of DNA to micrograms, more than enough for NGS. Phi29’s high fidelity results in low rates of false positives and negatives in analysis, making it well-suited for identifying single nucleotide polymorphisms (SNPs) and other mutations. MDA is the most popular current method of amplification, but it can create non-uniform representation of genomic regions.
The newest approaches to amplifying DNA, such as MALBEC, aim to use elements of both PCR and MDA, while mitigating the drawbacks of both methods. Although these hybrid methods do improve uniformity, they also depend on PCR, and have some of the drawbacks that entails, including a higher rate of false positives and negatives.
Whichever method you choose, once you have your starting material amplified, generating a library, sequencing, and analysis are relatively straightforward, and not too different from bulk cell analysis.
Single-cell sequencing experts can help!
If you’d like support with your single-cell genomics application, get in touch with GE scientific support. Or learn more about GE’s solution for Phi29-based DNA amplification, GenomiPhi, which can solve common challenges when performing single-cell sequencing.