First few Article Sentences
Next generation sequencing (NGS), often referred to as massively parallel sequencing, is a relatively new technique in molecular diagnostics that has truly served as a “disruptive technology” in the area of cancer treatment. Prior to the availability of NGS, one small fragment of DNA could be sequenced per reaction, using a technique called Sanger sequencing, that at the time of its discovery was also quite revolutionary. In Sanger sequencing, terminator nucleotides are used randomly in a PCR (polymerase chain reaction) reaction to create fragments of differing lengths with a known base at the terminal end. The fragments can then be lined up to deduce the sequence of the entire region being targeted. In contrast, NGS can sequence millions of fragments simultaneously, by sequentially adding reversible terminator nucleotides to numerous fragments of DNA from different samples, throughout all regions of interest, in one reaction. This is accomplished by adding adapters to the ends of the DNA fragments (used to affix fragments to a fixed surface, to “barcode” individual samples within a larger reaction, and to prime the PCR reactions), and then adding the reversible terminator nucleotides one by one, measuring the addition, and then cleaving the “terminator” portion so that another nucleotide can be added.
In this way, the amount of sequencing data retrieved per reaction is increased by an almost inconceivable amount. This benefits cancer patients in two ways. The first is that we can examine many genes that may be relevant to the patient’s tumor simultaneously, with a high level of accuracy, using only a small amount of tissue and for a reasonable cost. The second clear benefit is that we can look at these regions in replicate, typically hundreds to thousands of sequences from each individual targeted area, across all regions of interest. This is particularly important in cancer because tumor DNA is never 100% pure (there are always some stromal cells, inflammatory cells, or normal tissue cells included in the tumor, even after microdissection), and most solid tumors are known to be heterogeneous, meaning that an important mutation may only appear in a small amount of the total tumor DNA. It is much easier to identify a minor population of mutated DNA when there are thousands of sequences to compare, than when there is one tracing representing a single DNA sequence. The net result is a dramatic increase in the quality and quantity of information obtained.