How is whole genome sequencing actually done, and how is it different from whole

[Anonymous]

How is whole genome sequencing actually done, and how is it different from whole exome sequencing or a chromosomal microarray?

[Genetic Counselor]

To perform whole genome sequencing, a laboratory first extracts DNA from a blood or saliva sample and breaks it into millions of small, overlapping fragments. Unlike whole exome sequencing, this process skips the “capture” step in which chemical probes fish out only the coding fragments; instead, essentially all of the fragments, coding and non-coding alike, are read directly 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 each one. Because each stretch of the genetic code is typically read several times over from different overlapping fragments, a computer can piece the fragments back together and compare the result to a standard reference sequence, flagging places where a person’s genetic code differs from what is typically expected. Those differences, called variants, are then filtered and reviewed by geneticists to decide which, if any, explain the person’s symptoms.

Skipping the capture step is what sets whole genome sequencing apart from its two closest relatives. Whole exome sequencing uses that capture step to focus exclusively on the coding exome, which makes up only about 1 to 2 percent of the total genetic code; this keeps the volume of data far smaller and the cost lower, but it means the test is blind to the roughly 98 percent of the genetic code lying outside the exome, including the regulatory switches that control genes, deep intronic regions, and many kinds of structural rearrangements. Chromosomal microarray, a different technology altogether, does not read the sequence of DNA letters at all; it instead measures the amount of genetic material present across the genome, which makes it well suited to detecting a missing or extra stretch of DNA but blind to a single altered letter within a gene.

Because it reads virtually everything, whole genome sequencing can detect several categories of genetic change that whole exome sequencing is not designed to see well: structural rearrangements such as inversions, disease-causing changes in regulatory and splicing-relevant regions outside the exome, certain repeat expansion disorders, and, because it is not limited by capture probes, at least some sequence from the small separate genome carried inside mitochondria, the cell’s energy-producing structures. This last point comes with a caveat: detecting mitochondrial changes present in only a small fraction of a person’s cells, a situation called heteroplasmy, at very low levels can still require dedicated mitochondrial-specific testing performed at much greater depth than a standard whole genome sequencing run provides.

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