How does NGS actually read my DNA, and how is it different from other tests?

[Anonymous]

How does NGS actually read my DNA, and how is it different from karyotyping, chromosomal microarray, or older single-gene sequencing?

[Genetic Counselor]

To perform next-generation sequencing, a laboratory first extracts DNA from a blood or saliva sample and breaks it into millions of small, overlapping fragments. Each fragment is chemically prepared and then read by a sequencing machine, which determines the exact order of the four chemical building blocks, represented by the letters A, T, C, and G, that make up that fragment. Because millions of fragments are read simultaneously rather than one at a time, and because each stretch of the genetic code is typically read many times over from different overlapping fragments, a computer can piece the fragments back together with high confidence and flag places where a person’s sequence differs from what is expected. Those differences, called variants, are then filtered and reviewed by geneticists to decide which ones are relevant to the person’s symptoms.

This is fundamentally different from the older chromosome-based tests used in genetics. A karyotype photographs all forty-six chromosomes under a microscope and can spot only large, visible rearrangements, missing pieces, or extra pieces, generally above five to ten million building blocks in size. Chromosomal microarray improves on this considerably, scanning the entire genome at once for smaller stretches of missing or duplicated material, down to around fifty to one hundred thousand building blocks, but it only measures the amount of genetic material present. Neither test reads the actual sequence of letters within a gene, so neither can detect a single altered letter that changes how a protein is made, which is the cause behind the great majority of single-gene genetic conditions.

Next-generation sequencing fills exactly this gap. It cannot generally detect a genetic change involving a rearranged but balanced piece of a chromosome, and whole exome sequencing in particular has limited ability to detect large missing or duplicated stretches of DNA, which chromosomal microarray remains better suited to finding. For this reason, next-generation sequencing is usually thought of as complementary to, rather than a replacement for, chromosomal microarray and, in some situations, karyotyping; a geneticist decides which test, or combination of tests, fits a family’s specific situation best, and more than one test is often used together to build a complete picture.

[Author]

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