Genomics: DNA and RNA sequencing by NGS, cloning and qPCR

Definition of genomics and its applications in biotechnology

Genomics is the science that studies the genome of living things. Genome studies are used to map and study genes and their functions, including the identification of genes responsible for diseases.

Genomics uses a combination of recombinant DNA, DNA sequencing methods and bioinformatics to sequence, assemble and analyze the structure and function of genomes. It differs from "classical genetics" in that it considers all the hereditary material of an organism, rather than one gene or gene product at a time.

New technologies have literally allowed genomics to explode: Next Generation Sequencing (NGS) to map genomes, genome editing techniques such as CRISPR-Cas9, quantitative technologies to measure gene expression, using qPCR and digital PCR (dPCR).

What are the advantages of using a provider for genomic testing?

Access to the latest technologies and expertise

Saving time in the experimentation phase

Discover genomics services and exchange with the best providers

CRISPR-Cas9 and Genome Editing

Since the first publication of the CRISPR mechanism in 1987 by a team from Osaka University, the gene editing method known as CRISPR/Case 9 has come a long way.

What is CRISPR/Case, how does it work, and what are the steps to do genome editing with this tool?

CRISPR corresponds to a very particular type of DNA sequence of one of the Escherichia coli genes: It consists of 5 short repeated sequences, separated by short unrepeated sequences ("spacers"), hence its name: "clustered regularly inter-spaced short palindromic repeats", or CRISPR.

The Cas genes code for a family of DNA-cutting enzymes. It was described in 2008 that bacteria integrate DNA from viruses at the site of these spacers. As a result of this integration, bacteria acquire the ability to recognize and guide the Cas enzyme to cut viral DNA, and thus deactivate it. In 2012, a team will publish in Science how the CRISPR-Cas9 system can be used to cut any DNA.

The steps for using the method are as follows:

  1. Choose be modified (e.g., select the gene to be modified) and obtain its sequence the gene to,
  2. Select the endonuclease to be used: Cas9, Cas12, Cpf1, etc.,
  3. Construct the RNA Guide (gRNA) using tools such as Benchling,
  4. Assemble the guide vector in silico, choosing the most appropriate vector, e.g. a vector that has already been used to edit a gene,
  5. Cloning the guide vector containing: a backbone plasmid, the Cas9 gene, the sequence for the gRNA,
  6. Insert the vector into the cells to be modified, by transfection, transformation, or viral infection.
The future of technology clearly lays towards process improvement. The technology is still young, both technically (many cases have shown the limits in terms of specificity) and ethically (manipulations on human chimeric embryos are subject to licensing). Genetic engineering actors are developing the next versions of the CRISPR/Case system.

How does Next-Generation Sequencing (NGS) work?

Today, research instruments and reagents allow us to sequence entire genomes, whether known or named, to focus on regions of interest or to sequence the exome of isolated cells.

How did the sequencing technique developed in the 1970s by the British biochemist Frederick Sanger evolve to enable these advances?

The answer lies in the massively parallel technology offered by Next Generation Sequencing (NGS) and the phenomenal advances in bioinformatics.

Second-generation DNA sequencing is based on parallel amplification. DNA molecules are amplified in an emulsion PCR (emPCR). Adapter ligation and PCR produce DNA libraries with the corresponding 5′ and 3′ ends, which are immobilized on individual oligonucleotide-labeled microbeads. The bead-DNA conjugates can then be emulsified into emulsion droplets containing a single bead.

Clonal amplification then occurs during emPCR, with each template DNA being physically separated from all others, with daughter molecules remaining bound to the microbeads. Alternatively, PCR amplification can produce bridges and form clusters of clonal DNA populations in a solid phase reaction. The libraries produced by these two methods can then be read in a highly parallelized manner: the microbeads produced by emPCR can be washed on a picotiter plate, containing wells large enough to hold a single bead. Flow cell products from bridge amplification can be visualized by detecting fluorescent reversible terminator nucleotides at the ends of an ongoing extension reaction.

Third-generation sequencing is based on: single molecule sequencing (SMS single molecule sequencing), real-time sequencing, or any other feature that varies from second-generation sequencing. Two examples of mecanisms:

  • Nucleotide detection is done in a Zero Mode Waveguide (ZMW), as in the PacBio sequencers. DNA polymerase molecules are attached to the bottom of each ZMW, and target DNA and fluorescent nucleotides are added. Since the diameter is narrower than the wavelength of the excitation light, the illumination decreases rapidly up the ZMW: only the nucleotides incorporated during polymerization provide bursts of real-time fluorescent signal.
  • DNA sequencing of nanopores as used in the MinION sequencer from Oxford Nanopore Technology. Double-stranded DNA is denatured by an enzyme that passes one of the strands (ssDNA) through a biological nanopore embedded in a synthetic membrane, through which a voltage is applied. As the ssDNA passes through the nanopore, the different bases prevent ion flow in a distinct manner, making it possible to deduce the sequence of the molecule by monitoring the current at each channel.

Types of providers

Two major categories of genomics service providers:

Academic platforms

Academic platforms are solicited for sequencing that requires the development or adaptation of a method. As sequencers are very expensive, academic laboratories may form a group to finance them. The service is provided by a technological platform that made itself available from external structures such as research institutes and companies.

The servicers

Genomics service companies offer services tailored to the demands of private or public clients: sequencing, genome editing, microarray, method development. They can be highly specialized (on a technology or a type of application), or generalist, and can validate methods such as qPCR, using GLP (good laboratory practice) principles.

The post-genomic era

The genomic era is a period that stretches from the discovery of DNA by Watson and Crick to the sequencing of the entire human genome. This project, which began in the late 1980s and ended in 2003, aimed to provide answers to all the questions related to the functioning of our cells.

Far from having met these expectations, the project brought to light a higher level of complexity regarding the mechanisms that regulate the behavior of our cells and the expression of our genes. The era of the genomic post then appeared, bringing with it all the "omics" science groups: proteomics, metabolomics, transcriptomics etc.

Science in the post-genomic era is therefore concerned with determining when and under what conditions a gene is expressed. These studies aim to understand the organization of gene networks, the DNA/DNA, protein/DNA or protein/RNA interactions that lead to the expression of these genes. This era has also allowed the emergence of Bioinformatics, whose aim is to model in silico the interaction networks to lead to a virtual living cell.

The technologies used

  • Molecular cloning techniques
  • Nucleic acid extraction
  • PCR, qPCR, dPCR
  • Sanger and NGS sequencer
  • Microarray
  • CRISPR/Cas9 and other gene-editing technologies

Estimated rates for this type of service

Sequencing typically varies according to the size of the sequence and can range from a few tens of Euros (Sanger sequencing) to a few thousand (NGS on large genomes).
Quantitative PCR requires a development phase of between €3,000 and €10,000 per gene, depending on the method used (probes vs fluorophore such as SYBR® Green; simplex vs multiplex, qPCR vs dPCR).
The modification of a cell line by CRIPSR/Cas9 is estimated between €5,000€ - €10,000.

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