Rapid genomic analysis offered by next generation sequencing (NGS) is ideal for personalized medicine approaches to clinical genetics, microbiological profiling, and diagnostic oncology. Many standard clinical samples are preserved as formalin-fixed, paraffin-embedded (FFPE) tissues, which presents obstacles for use in NGS analysis.
FFPE tissue preservation has the benefit of keeping samples intact for histological examination for decades. However, it comes at a price — chemical crosslinks can diminish the quality of DNA needed for downstream analysis. Extraction of FFPE-DNA suitable for NGS analysis requires conditions that can effectively overcome the nucleic acid crosslinking caused by formalin fixation without causing further DNA damage. Beyond that, extracted DNA requires controlled shearing into manageable fragments for NGS library preparation.
DNA Extraction and Shearing in One Seamless Protocol
The best NGS experiments start with a robust FFPE-DNA extraction platform and shearing of extracted DNA into well-defined fragments. Until recently, DNA extraction and shearing were performed as separate and distinct protocols. This led to loss of material and a lack of continuity or standardization between experiments. There had to be a better way. Enter the combined, seamless DNA extraction-shearing platforms, like the FFPE DNA Extraction Kit and the Bioruptor® from Diagenode.
The advantages of this combined extraction-shearing protocol are compelling:
- Gentle DNA extraction – One concern with DNA extraction from FFPE tissues is that harsh conditions for deparaffinization and dehydration cause additional damage to the DNA. This concern is put to rest with the DNA Extraction Kit. The paraffin removal and rehydration processes are combined into one step that circumvents the use of harsh solvents or excessive temperatures. These mild treatments undo crosslinks created by formalin fixation, while preserving DNA integrity.
- DNA shearing is incorporated into the DNA extraction protocol – Shearing of DNA into a consistent range of fragment sizes is an essential prerequisite for successful NGS library preparation. By coupling the kit and the shearing instrument into one protocol, DNA extraction and shearing processes are consistent between experiments and carefully optimized to result in high-yield, NGS-quality DNA fragments for reliable downstream analysis.
- High throughput – A busy lab may have many samples in the queue for FFPE-DNA extraction. The protocol allows multiple samples to be processed simultaneously for maximum efficiency. For example, the Bioruptor® can shear 12–16 DNA samples at once, depending on the model.
Efficient DNA Shearing and Quality Control
This seamless protocol is performed in just a handful of steps with minimal manipulation of the material.
Perform deparaffinization of DNA in microtubes containing 10?M thick tissue sections by incubating in FFPE Extraction buffer. Vortex and sonicate using the Bioruptor®, then move the supernatant to a new tube. Treat the DNA with Proteinase K and incubate with Cross-link removal buffer to remove contaminating proteins. Isolate the DNA using column filtration to complete the DNA extraction protocol. To fragment the DNA, use one of the standard Bioruptor® protocols to obtain your desired fragment size for DNA sequencing. Keep in mind that the desired DNA fragment length for NGS depends on the commercial platform.
After successful extraction and shearing, assess the DNA for quality, yield, and fragmentation state, prior to NGS library preparation.
There are a few ways to accomplish this:
- Resolve the sheared DNA with agarose gel electrophoresis. This is a low-tech way to visualize DNA integrity. Discrete banding patterns indicate consistently fragmented DNA, while smears indicate DNA degradation.
- Use microfluidic capillary electrophoresis of DNA to visualize fragmentation patterns and to confirm the desired size of DNA fragments. DNA sizing and quantitation measurements are based on comparisons with internal standards.
- Perform end-point PCR to assess the yield and quality of DNA.
Once the size-selected and fragmented DNA is validated, it is ready for NGS library preparation.
Overcome Traditional Obstacles with Using Older Samples
Traditional protocols can result in poor quality DNA from FFPE samples. This is because excessive fixation in formalin results in extensive crosslinking, which can be very difficult to reverse. Also, older FFPE samples, although preserved histologically, tend to release DNA of a lower molecular weight than fresh or frozen tissue.
However, combined DNA extraction-shearing platforms, like the FFPE DNA Extraction Kit with the Bioruptor can provide consistent, high yields of fragmented DNA. This continues to transform DNA sample preparation as the NextGen revolution advances from bench to clinic. You can find the full DNA extraction and shearing protocol here. For additional information on DNA extraction from FFPE-samples, shearing, and NGS, check out the resources listed below.
- Diagenode. FFPE DNA extraction protocol using the Bioruptor®.
- Lakha W, et al. (2016) DNA fragmentation and quality control analysis using Diagenode shearing systems and Fragment Analyzer. Nature Methods. 13(10).
- Harper K. How to Make Sure Your NGS Results are Robust.
- Seigel G. Kiss your samples goodbye: Outsourcing your Next-Gen experiment