Next Generation Sequencing (NGS) represents the latest evolution of sequencing. For many years, sequencing was done by capillary electrophoresis (Sanger sequencing). Capillary electrophoresis sequencing allows us to reconstitute the sequence of a single DNA fragment. Sanger sequencing is done by recording the signals of incorporation of fluorochromes-labeled nucleotides, which are used to synthesize the strand complementary to the original DNA fragment. Even NGS is based on a similar principle; however, the reaction may be done for many fragments of DNA in parallel, not just one. Through NGS it is therefore possible to obtain an enormous amount of sequences (in a single stroke you can get gigabases or terabases of information) more quickly and at a much lower cost. For this reason, NGS is also known as high-throughput sequencing.
In NGS, the DNA of an individual is broken into many small fragments (for example, through the use of ultrasound) to constitute the so-called sequencing library. These small fragments serve as templates for the synthesis of numerous, complementary fragments called reads. Each small fragment of DNA is copied many times in a variable number of reads. Depending on the desired level of precision, it is possible to set the system to achieve a certain level of coverage, i.e., a certain number of reads for each fragment of the library. For example, 30 reads per fragment, which would have been defined in jargon as 'coverage 30x', are already sufficient for routine diagnostics of Mendelian diseases, while the diagnosis of somatic mutations typical of tumors may require coverage up to 1000x. A computer then collects all the reads and aligns them with the reference sequence of the human genome annotated in the databases. By this way the reads can be reassembled like in a puzzle to obtain the sequence of the gene or of the entire genome.
In NGS, the DNA of an individual is broken into many small fragments (for example, through the use of ultrasound) to constitute the so-called sequencing library. These small fragments serve as templates for the synthesis of numerous, complementary fragments called reads. Each small fragment of DNA is copied many times in a variable number of reads. Depending on the desired level of precision, it is possible to set the system to achieve a certain level of coverage, i.e., a certain number of reads for each fragment of the library. For example, 30 reads per fragment, which would have been defined in jargon as 'coverage 30x', are already sufficient for routine diagnostics of Mendelian diseases, while the diagnosis of somatic mutations typical of tumors may require coverage up to 1000x. A computer then collects all the reads and aligns them with the reference sequence of the human genome annotated in the databases. By this way the reads can be reassembled like in a puzzle to obtain the sequence of the gene or of the entire genome.
NGS machines available today are produced by many different brands and they are very flexible devices. A NGS sequencer can actually be used for different types of applications:
1. Whole-genome sequencing (WGS) - also known as whole-genome shotgun, this is the analysis of the entire genome of an individual.
2. Whole-exome sequencing (WES) - analysis of the entire coding region of all the genes of an individual.
3. Targeted sequencing - analysis of a group of genes (panel) or of a single gene.
4. Transcriptome analysis - analysis of all RNA produced by a cell (transcriptome).
1. Whole-genome sequencing (WGS) - also known as whole-genome shotgun, this is the analysis of the entire genome of an individual.
2. Whole-exome sequencing (WES) - analysis of the entire coding region of all the genes of an individual.
3. Targeted sequencing - analysis of a group of genes (panel) or of a single gene.
4. Transcriptome analysis - analysis of all RNA produced by a cell (transcriptome).
Programs 2 and 3 require an additional step (target enrichment) and can also be performed for many samples simultaneously through the so-called technique of multiplexing. The DNA of each individual can, in fact, be distinguished by attaching a specific sequence (barcode sequence) to it. Since barcode sequences are also sequenced, the reads belonging to each individual can be recognized and sorted before alignment thanks to the barcodes.
