What tools do we have?
The molecular diagnostic tools underwent a major revolution in the last two decades. That said some of the old favorites did not go completely away, we still use some of them; RFLPs, Southern blots or SSCPs just to mention a few. Why some of the methods do not go away completely despite alternatives being present? The laboratories are used to them, they are simple, reliable and the volume of samples is not high enough to invest in a more expensive, modern technology.
However, those methods while using the genetic information did not report sequence back. Development of DNA sequencing technology was critical for further advances in molecular diagnostics. The Sanger method using dideoxynucleotides have allowed us to sequence DNA relatively easily. While we started with awkward radioactive slab gels, we soon moved into using fluorescent dye terminators and fully automated sequencers.
The invent of PCR was thought to dramatically change the diagnostic landscape overnight. And while it did, the change was definitely not overnight. The initial problem was the high cost of reagents, second problem was reliability of the technique in early days. A lot of work have led to development of modifications of PCR, such as end-point real time PCR and quantitative real time PCR.
Further improvements in technology led to development of microarray technology. It was initially used for expression profiling, then became used for SNP, CNV and cytogenetic use. Affymetrix technology also became useful for sequencing by hybridization. However, this was not the end of development. More recently next generation sequencing seems to be coming of age and it has moved from research technology to a clinically-ready tool.
What tools to choose?
One can feel a little intimidated with the choices available and all the companies trying to sell their products as the best thing since sliced bread. It takes a lot of laboratory skills and experience together with genetic knowledge to identify the best solutions and assess them for suitability in a diagnostic laboratory.
Let’s consider a simple example of carrier screening for a 100 of genetic diseases, among them cystic fibrosis, Tay-Sachs, Gaucher, beta-thalasemia, SMA, or fragile X. What tools to use? Is there a single validated testing platform that will identify all of them? There are some tests that cover at least a part of the set of those diseases, Luminex assay (xTAG Cystic Fibrosis Kit), NGS panels (MiSeq CF, MiSeq carrier screening assay), MLPA assays, Affymetrix-based resequencing assays (TessArae), and a lot of single gene PCR and/or Sanger sequencing tests.
Is there an advantage to a particular assay compare to another? Yes, sequencing assays deliver more information than a PCR test focused on a single mutation. They can also identify novel mutations, which is considered an advantage as well as a problem. Should novel findings be reported or not, they might be clinically unimportant? More findings create a need for more confirmation tests, thus being more expensive. PCR based assays are quicker, cheaper and deliver a Yes or No answer for a particular mutation, and there are no accidental findings. How do the sequencing technologies compare? Targeted sequencing for carrier screening is now provided by NGS and has a clear advantage over a resequencing technology on arrays. NGS can identify deletions, insertions or indels, which cause difficulty for hybridization-based sequencing. It can seem a little excessive to use NGS for carrier screening, but NGS can test many more conditions than microarrays and it is not dependent on what mutations were targeted by the assay.
Carrier screening is only the one part of genetic diagnostics efforts. We also diagnose patients presenting with a disease after birth, “fishing” so to speak for a cause of the disease. Additionally, tests are run for complex diseases (e.g. neurodegenerative disorders), or disease predisposition (e.g. cancer). Those kinds of assays need to be able to screen multiple genes, identify “de novo” mutations and in such cases WGS or whole exome sequencing is being a preferred method to speed up the analysis. In some cases amplification panels are used to make analysis simpler.
It seems that we are going to see changes in diagnostic tools used in clinic but at the same time it also appears that old methods are not going to be discarded from the labs just yet. It is important to remember that as much as we want to have a one test for all diseases, that is not always possible. A more important thing is to choose the best tool for the job. If you are a service lab you need to do your homework and bring in new technology that is the best for the tests you want to run. On the other hand, if you develop tests you need a very good understanding of customers’ needs and genetics. We all need to work together to match the tools to the needs we have.
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