Massive Sequencing

General Description

Massive sequencing, or next-generation sequencing (NGS) as it is commonly known in English, is a term that encompasses various procedures used to determine the sequence of DNA nucleotides. The objective is to analyze genes and correlate them with the development of diseases or disorders. Its primary application is clinical diagnosis.

The most significant advancement in massive sequencing is the ability to analyze a large number of DNA sequences or specific genes in parallel. The patient's genetic material is compared with a similar phenotype to determine the presence or absence of alterations. If the result is negative, it must be assessed whether there is sufficient justification to expand the study through exome sequencing or whole genome analysis.

The most commonly used techniques in massive sequencing include:

• Sequencing by synthesis or polymerization: Useful for analyzing small DNA fragments. In these processes, a DNA polymerase enzyme, which plays a role in DNA replication, is used as a template.
• Real-time sequencing: A very rapid process since it does not require stopping the division to incorporate a new base or perform a reading. A device is used to restrict the field of view to newly added nucleotides in order to record the fluorescent signal.

The ability to provide increasingly precise diagnoses and relevant information for tailoring treatments to each patient’s specific characteristics makes NGS one of the cornerstones of personalized medicine.

Massive sequencing began developing in the 1970s, although its widespread use did not occur until the early 21st century. It is, therefore, a continuously evolving process that is expanding and finding new applications.

When Is It Indicated?

Massive sequencing has a wide range of applications and is indicated in cases such as:

• When there is suspicion that a patient has a genetic disease that can be diagnosed through molecular genetics processes.
• In oncology patients, to detect mutations involved in cancer development, allowing for personalized treatments.
• In the microbiological diagnosis of infectious diseasesInfectious DiseasesInfectious diseases .
• For individual identification in forensic medicine.
• In transplants, to determine HLA typing and improve donor-recipient compatibility.
• In prenatal diagnosis.

How Is It Performed?

Each method used in massive sequencing follows specific procedures. The most prominent include:

• Sequencing by synthesis or polymerization:
• Ion-conductance sequencing: Identifies pH changes during DNA synthesis. This is achieved by adding nucleotides (molecules consisting of a nitrogenous base, a phosphate group, and a simple sugar molecule), which release a proton and alter the hydrogen potential. This process is repeated multiple times by adding one type of nucleotide at a time to allow identification.
• Reversible terminator sequencing: Uses fluorescently labeled terminator nucleotides. The labeled molecules are not removed until the fluorescent signal is recorded, and only then is the next nucleotide added.
• Real-time sequencing:
• Single-molecule DNA sequencing: Involves adding labeled nucleotides to a DNA strand while it is undergoing synthesis, forming new DNA molecules. A support structure containing wells is used, where DNA polymerases are anchored along with fragments of the DNA being studied, forming a closed circular chain. When labeled nucleotides are added to the well, fluorescence is activated, and the sequence of each fragment is obtained.
• Nanopore-based sequencing: Involves passing a DNA strand through a membrane protein to analyze changes in electrical current during the process. Using a device equipped with a specific algorithm, the nucleotide sequences are visualized.

The data obtained through these techniques are stored in different types of files by technological devices:

• FASTQ file: Stores raw data on DNA sequences and their quality in text format.
• SAM file: Displays sequence alignments, meaning it compares a fragment of the patient's DNA with a reference sequence.
• BAM file: Contains compressed data from the SAM file. To facilitate access, a genomic position index can be created for direct retrieval.
• VCF file: A more flexible format that can store DNA sequence variations relative to reference genes, along with specific annotations.

Risks

Massive sequencing does not pose health risks to patients. The main risk lies in the inability to analyze the requested sequence. Additionally, it only studies a portion of the genome, meaning there could be anomalies in other genes that are not included in the study.

What to Expect from Massive Sequencing

To conduct a genetic study using massive sequencing, a patient sample is taken—most commonly a small amount of peripheral blood. The extraction procedure follows the same steps as routine blood tests:

1. The arm is extended with the fist clenched.
2. A tourniquet is placed above the elbow.
3. A needle is inserted into a vein in the back of the elbow, causing a slight prick sensation.
4. The hand is opened, and the tourniquet is released.
5. The blood is collected in a lavender-top tube containing EDTA (ethylenediaminetetraacetic acid), an anticoagulant that keeps the sample in liquid form for a longer period.

Depending on the sequences analyzed and the process performed, results may take approximately two weeks. These results are classified into five categories:

• Pathogenic variant (PV)
• Likely pathogenic variant (LPV)
• Variant of uncertain significance (VUS)
• Likely benign variant (LBV)
• Benign variant (BV)

A specialist must evaluate the results to determine the next steps.

Specialties That Request Massive Sequencing

Massive sequencing is a technique used in genetics to support the diagnosis of conditions primarily related to oncology, microbiology, and internal medicine, among other specialties.