The finished Human Genome Project together with the advance of Next Generation Sequencing (NGS) technologies, led to what we call “Post Genomic Era”. In a short time and for an affordable price, it became possible to have all or some parts of the DNA sequenced and easy access to several databases that provide hundreds of genetic mutations.
However, it is important to remember that there are differences between NGS techniques, and that they are not suitable for diagnosing all types of genetic disorders.
My family has Breast Cancer history, can I take a test for Exoma? My grandmother discovered a pathogenic mutation, how can I test myself?
Before explaining what NGS is, read a brief introduction about the first generation DNA sequencing.
DNA sequencers are devices that read a DNA sample and generate an electronic file with symbols that represent the sequence of nitrogenous bases (A, C, G, T) present in the sample. In other words, the DNA sequencing is the determining of the exact order in which the nucleotides are found.
The first popular method of DNA sequencing was chain termination, published in 1977 by Frederik Sanger, and has been used by research centers around the world to this day.
The Sanger sequencing is a useful and effective “first generation” technology in sequencing 500-900 base pair DNA fragments, being specifically used to sequence small portions of DNA, such as bacterial plasmids or DNA fragments amplified by PCR (polymerase chain reaction or polymerase chain reaction).
In 1986 the first automatic DNA sequencer, ABI 370, was launched, and in 1998, the first capillary electrophoresis sequencer, ABI 3700.
With automation, it was possible to carry out large sequencing projects, such as the complete Human Genome Project, costing billions of dollars and using large centers with a lot of machines installed.
Despite the success in sequencing the complete human genome, Sanger sequencing is an expensive and inefficient method for large-scale projects. The new NGS techniques are more efficient and less expensive for tasks like that.
So, what is the Next Generation Sequencing (NGS)?
Next-Generation Sequencing, also called Massive Parallel Sequencing or High Performance Sequencing are different terms that refer to a group of different and modern DNA sequencing methodologies (Figure 1).
NGS can be characterized as automated, parallel and high-throughput sequencing.
The arrival of NGS technologies on the market has changed the scientific approaches in basic, applied and clinical research and in genetic diagnosis, bringing new perspectives for precision medicine. These technologies allow DNA to be sequenced much more quickly and cheaply compared to Sanger sequencing, revolutionizing the study of genomics and molecular biology.
The NGS sequencers are different, but they are all based on massive parallel processing of DNA fragments. That is, while a capillary electrophoresis sequencer processes a maximum of 96 fragments at a time, next generation sequencers can read up to billions of fragments at the same time.
With NGS, you can sequence the whole genome or just specific areas of interest, including all of the approximately 20,000 coding genes (Whole exome sequencing or WES) or a small number of individual genes (Targeted sequencing). In both, the NGS can detect mutations in the DNA sequence that can be small alterations (substitutions), insertions and deletions of nucleotides.
The result of NGS is a file with the exact position of each DNA nucleotide. From it, is possible to analyze alterations in the individual’s DNA sequences. In this context, there are several online platforms that estimate the possible damage that a specific alteration can cause in the protein it encodes.
Each platform makes this prediction through specific metrics and thus classifies whether the variation is more likely to have not caused any alteration in the protein (that is, it is a benign mutation) or to have caused a mild or severe alteration (possibly pathogenic or pathogenic mutation).
In addition, NGS is highly efficient in the molecular diagnosis of genetic diseases and hereditary or non-hereditary cancers caused by germline variants, that is, genetic alterations that have been inherited and therefore affect all cells of an individual. And somatic variants, mutations that occurred after development, at some point in life, and are present in one or more specific tissues.
What are the NGS types?
There are basic differences between the type of NGS that may be required for research or clinical diagnosis. These differences are not necessarily based on the platform used to perform the sequencing, for example MiSeq Illumina or Ion Torrent, but on the sequences resulting from the sequencing. Therefore, it is necessary to know what to look for at the end of the sequencing before requesting the type of NGS.
In the following topics we will explain what each type of NGS is and what each one is indicated for.
Whole Genome Sequencing (WGS)
The sequencing of the complete genome is, as the name says, the sequencing of all the genetic material or DNA of an individual. This sequencing will include both coding and non-coding DNA. The WGS results in a very large file with a lot of information about the genetic material that may have a clinical interpretation or not.
Approximately 2% of alterations in the coding portion of DNA are more susceptible to clinical interpretation, whereas alterations in approximately 98% of DNA do not yet have information about its possible consequences for the individual.
In addition, WGS has a higher cost for both processing and storing the result. Therefore, is more indicated to study the consequences of alterations in regions that have not yet been studied or the search for alterations that cause not elucidated diseases, for example.
Sequencing the complete exome (WES)
WES is the sequencing of the coding portion of DNA, that is, of all the DNA sequences that encode the approximately 19,000 genes in the human genome.
The term exome refers to the set of exons, which are the DNA sequences that will pass through the transcription and translation steps until they are encoded in proteins.
Alterations in genes cause changes in proteins and are therefore more likely to cause disease. Exome mutations are responsible for the vast majority of genetic diseases.
Thus, WES is an efficient laboratory test and most suitable for clinical diagnosis, especially in cases where there is a diagnostic hypothesis of more than one specific disease, or even if there is no specific diagnostic hypothesis.
The result of a WES is a file with the mutations found in all the individual’s genes, which requires a longer analysis time.
Sequencing of target genes, targeted sequencing or gene panel
The sequencing of target genes or gene panel is the NGS performed from only one group of genes of interest.
This type of sequencing is similar to WES, however, instead of sequencing all of the individual’s genes, the sequencing is performed only in some specific genes: already associated with one or more diseases, for example. Therefore, when there is a diagnostic hypothesis for a specific disease, the gene panel is more recommended than WES in clinical diagnosis.
Currently, laboratories that realize NGS provide panels of genes associated with several genetic diseases, such as breast cancer and other cancers, skeletal, muscular diseases or leukodystrophies, for example.
The benefits of gene panel sequencing over WES are the savings in the cost of NGS, since fewer genes will be sequenced, and decreased analysis time.