Genomic and Sequencing Technologies
NGS (Next-Generation Sequencing)
Development of Next-Generation technologies revolutionizes sequencing in terms of produced sequences, costs and analytical skills.
The new generation of high-throughput sequencers raised the amount of data obtained with a single run. They increased the number of applications and fields, and for some cases, replace the microarrays. The limitation is no longer data generation but their storage and analysis.
NGS (Next-Generation Sequencing) technologies are based on fixed DNA molecules onto solid support, cyclic sequencing reactions and detection by imaging. The process can be summarized with the following steps:
- Sample preparation (amplification, adapters addition) to create a library.
- Sample fixation on the glass slide and generation of the clusters;
- Cyclic sequencing reactions with a fluorescent emission for each type of nucleotide;
- Base calling;
- Data filtration, analysis and interpretation.
Several samples can be sequenced at the same time, with a great depth. Millions of reads are produced for each sample; each base is sequenced several times (10X, 20X…).
Applications are numerous: variation discovery, genomic rearrangement identification, gene expression profiling, whole genome sequencing, capture for target re-sequencing or DNA methylation studies.
DNA microarray have proven very useful in many life science areas. They provide high throughput information to biologists and give a panoramic view of the cell transcriptional status: this technology allows expression measure of tens of thousands of genes simultaneously by comparing their relative levels in the different experimental conditions. We can then identify differentially expressed genes, co-regulated genes or pathways of interest.
Oligonucleotides are synthesized on a solid surface. These probes are gene specific and hybridize with labelled cRNA samples. During the scan, the dyes are excited; the fluorescence emission is detected and quantified. The intensity differences reflect transcript variation for the experimental conditions.
Array CGH (Comparative Genomics Hybridization)
Array CGH is a molecular cytogenetic technique which combines microarray technology with the CGH approach. Metaphase chromosomes are replaced by oligonucleotides and resolution is higher. This technology allows a precise mapping of the regions with aberrations. It is an essential diagnostic tool to detect chromosomal copy number variations. Array CGH is able to detect unbalanced chromosomal abnormalities.
For example, this technology compares a patient’s genome against a reference genome and identifies the differences (amplifications and deletions).
Custom arrays can be designed to target regions of interest (genes, chromosomes…) on any type of species.
Array SNP Genotyping
An SNP array is used to detect polymorphisms within a sample. The principles are the same as the DNA microarray: a hybridization step, a technology based on fluorescence, a solid surface, oligonucleotide probes and a system to interpret signals.
High density SNPs arrays allow testing a large number of polymorphisms in the same study. The applications include whole-genome genetic linkage analysis, identification of genetic abnormalities in cancer for prognostic or diagnostic uses.
Quantitative real-time PCR (RT-qPCR) is based on the ability to track in “real time” the process of PCR using fluorescence. Several technologies are available for detection:
- Non-specific fluorescence dye that intercalate with double-stranded DNA;
- Specific DNA probes labelled with fluorescent reporter. In this case, the intensity is directly proportional to the amplified target molecules (technology Taqman).
Data are collected at each PCR cycle and represent the amount of product amplified to that point. During the first cycles, the intensity is very weak and will allow defining the baseline. The accumulation of PCR products causes a measurable change in the intensity. When the fluorescence exceeds the threshold (exponential phase), the Ct (Threshold Cycle) is defined: this value is used to quantify molecules.
RT-Q PCR brings two types of quantitative answers :
- Absolute quantification: it gives the exact number of targets by comparing standards using a calibration curve.
- Relative quantification: this method is based on reference genes (housekeeping genes) to determine fold-differences; it is easier to use as the calibration curve is not necessary.