Next-generation sequencing (NGS) is a way of finding out the order of the letters in DNA or RNA, the genetic material inside our cells. DNA and RNA are made of four different letters: A, C, G, and T for DNA, and A, C, G, and U for RNA. The order of these letters determines what traits a person has, such as eye color, blood type, or disease risk.

NGS works by breaking up the DNA or RNA into many small pieces and reading the letters on each piece. Then, it uses a computer to put the pieces back together and compare them to a reference sequence. This way, it can find out what is different about the DNA or RNA of a particular sample.

NGS is used in pathology to help diagnose and treat diseases that are caused by changes in the DNA or RNA. For example, some cancers are caused by mutations (small changes) or rearrangements (large changes) in the DNA of certain genes that make the cells grow out of control. After examining a tissue sample under the microscope, a pathologist may perform NGS to narrow down or confirm the diagnosis. NGS is often combined with other types of tests such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), or flow cytometry.

NGS can also be used to select drugs that can target the changes identified in the tumour. This is often described as precision medicine because it helps provide the right treatment to the right patient at the right time. NGS can also help find out how a person responds to drugs based on their DNA or RNA. For example, some people have variations in their DNA that make them metabolize drugs faster or slower than others. This can affect how well the drugs work or how likely they are to cause side effects. NGS can help identify these variations and adjust the dose or type of drug accordingly. This is called pharmacogenetics or pharmacogenomics because it helps personalize the drug therapy based on the genetic makeup of the person.

What are some of the most common genetic changes tested for by next-generation sequencing?

NGS can be used to look for thousands of genetic changes. However, at present, only a small number of genes are commonly assessed.

Genetic changes commonly assessed by NGS include:

• EGFR, KRAS, BRAF, and PIK3CA: These are genes that are involved in cell growth and division, and they can be mutated or changed in some cancers. Testing for these genes can help diagnose the type and stage of cancer, as well as guide the choice of treatment. For example, some drugs can target cancers that have mutations in EGFR or BRAF1.
• NTRK, ROS1, and ALK: These are genes that encode proteins called tyrosine kinase receptors, which are also involved in cell growth and division. Some cancers have rearrangements or fusions of these genes with other genes, which can make the cells grow out of control. Testing for these genes can help identify cancers that can be treated with drugs that block the activity of these receptors.
• CFTR, PAH, GALT, and HBB: These are genes that are associated with inherited disorders that affect different organs or systems in the body. Testing for these genes can help diagnose or screen for these disorders, as well as provide information about the risk of passing them on to children. For example, CFTR is the gene that causes cystic fibrosis, a disease that affects the lungs and digestive system; PAH is the gene that causes phenylketonuria, a disease that affects the metabolism of an amino acid called phenylalanine; GALT is the gene that causes galactosemia, a disease that affects the metabolism of a sugar called galactose; and HBB is the gene that causes sickle cell anemia, a disease that affects the shape and function of red blood cells.
• BRCA1 and BRCA2: These are genes that are involved in DNA repair and protection from damage. Mutations or changes in these genes can increase the risk of developing breast, ovarian, prostate, or pancreatic cancer. Testing for these genes can help identify people who have a higher chance of developing these cancers, as well as provide options for prevention or early detection.
• EWSR1, FUS, and SS18: These are genes that encode proteins called transcription factors, which are involved in regulating gene expression. Some sarcomas have fusions or rearrangements of these genes with other genes, which can affect the differentiation and development of the cells. Testing for these genes can help classify the type and subtype of sarcoma.
• PDGFRA, PDGFRB, and KIT: These are genes that encode proteins called platelet-derived growth factor receptors, which are also involved in cell growth and division. Some sarcomas have mutations or amplifications of these genes, which can make the cells more responsive to growth signals. Testing for these genes can help identify sarcomas that can be treated with drugs that inhibit these receptors.