Featuring: Dr Rachel Butler, Consultant Clinical Scientist, University Hospital of Wales
In the previous film, we showed you the journey of the tumour sample through the histopathology lab. Here, we will discuss the next stage: extraction of DNA from solid tumour tissue. Extraction of DNA from fresh frozen tissue is the preferred method and usually yields good quality samples for whole genome sequencing. However, the quality of DNA obtained from formalin-fixed, paraffin-embedded tissue is often poor and, as a result, not of the required standard for whole genome sequencing. Genomics England is actively working to improve the methods for extraction of DNA from FFP samples, so always check you are using the most up to date specification.
Dr Rachel Butler: Histopathologists can actually look at those sections down a microscope and can see which parts of the tissue contain tumour and don’t contain tumour. Those sections get passed onto the molecular laboratory with the sections of the tissue actually having been highlighted as to which bits of the tissue are for DNA extraction, and they get physically highlighted on those glass sections on the slide and we can actually start performing our DNA extraction.
FFPE specimens need to be treated prior to extraction to remove the paraffin and rehydrate the tissue. This can be achieved with solvents such as xylene or by an ultrasonicator, such as the Covaris shown here. The tissue is digested with Proteinase K, then heated to reverse crosslinks and the resulting lysate is purified using a spin column. The alternative method using the QIAamp FFPE tissue kit has many steps in common with this method, but the paraffin is removed by solvents rather than by sonication. As always, be sure to follow the most up-to-date protocols issued by Genomics England.
On assessing the quantity of DNA extracted from FFPE tumour samples, we need to measure how well the DNA is able to be amplified. Currently this is done by performing real-time PCR and comparing the extracted DNA to a control sample. The comparative result is called the deltaCq value. The deltaCq value determines how much DNA is required for whole genome sequencing. These values will be continually reviewed and refined throughout the 100,000 Genomes Project but are currently as shown in the table. Samples with a higher deltaCq are regarded as unsuitable for sequencing. The deltaCq will be measured by the biorepository, but many laboratories are beginning to undertake their own deltaCq analysis. For the biorepository to have enough material to undertake CQ, you will need to send a minimum of 2 micrograms of DNA.
Dr Rachel Butler: The quality of the paraffin-fixed tumour samples is always going to be a problem. It has been a problem for a long time and it’s particularly a problem when it comes to performing DNA sequencing, particularly next generation sequencing.
The table here defines the current output specification for DNA extraction for whole genome sequencing from tumour.
Dr Rachel Butler: Every step is in there for a reason and is obviously going to be really vital, and if you deviate from that SOP, it is quite likely that therefore the quality or the quantity of the DNA is going to be sub-standard.
In this film, you have seen the recommended procedures and best practices for extracting DNA for whole genome sequencing from tumour samples. Following these important steps will greatly increase the chances of obtaining high-quality DNA to be sent to the biorepository and ensure the success of this important project.