Overview

Formalin-fixed, paraffin-embedded (FFPE) tissue from human biopsies are typically collected and preserved for morphological studies by pathologists and researchers. This fixation technique allows storage of the material at room temperature for many years by cross-linking the nucleic acids and proteins within the cells and encasing the tissue in wax. With advances in library preparation and next generation sequencing techniques, nucleic acids preserved in these samples may now be recovered to yield important genomic information correlated with outcomes. For this reason, archived FFPE tissue samples have become a rich and precious source of study material in cancer research and medicine. Increasingly, laboratories are utilizing FFPE samples to profile and identify disease-related genes, mutations, and biomarkers. Biobanks worldwide have tens of millions of such archived samples.

Workflow

In a typical workflow, genomic DNA is first isolated from the FFPE sample and then mechanically sheared to yield short double-stranded molecules. Due to the archiving process, DNA is usually present in limited quantities and in damaged states. Therefore, in order to produce a high quality library and to ensure the highest quality sequencing results, the choice of the library preparation method is critical. ThruPLEX® technology is the method of choice for many leading laboratories to construct libraries from FFPE samples because of its simple single-tube workflow, superior performance in repairing damaged DNA, and efficiency in amplifying low input amounts of DNA. The ThruPLEX technology is highly efficient, accurate, and does not require purification steps where DNA can be lost. Most importantly, its patented DNA repair and ligation chemistries enable sensitive and consistent sequencing from highly degraded DNA extracted from archived FFPE samples.

Picture1Technology

Step 1.ThruPLEX technology features improved repair chemistry ensuring the FFPE sample is prepared for ligation.

Step 2. Blunt-end ligation occurs with high-efficiency. Primer dimer formation and subsequent background is reduced since the hairpin adapters have blocked 5’ends and no single-stranded tails are present.

Step 3. Background is reduced by destroying unused adaptors after ligation. High fidelity amplification reduces errors introduced through amplification.

Overall, the time to complete the process is reduced by using compatible buffers and multiplexed enzymatic reactions that do not require
intermediate purifications.

Contact a Rubicon Genomics Application Specialist

Additional information is available in the Application note entitled “Use of ThruPLEX® DNA-seq Kit with FFPE Tissues”. Obtain your copy by registering here.

Learn more about ThruPLEX DNA-seq Kit

*Protected by US patent 7,803,550 and EP1924704