Formalin-fixed, paraffin-embedded (FFPE) samples are typically collected and preserved from human biopsies for histological studies. The formalin fixation method allows storage of the material at room temperature for many years by cross-linking the cellular contents of nucleic acids and proteins and encasing the sample in wax. Biobanks and researchers worldwide have millions of FFPE samples stored. With advances in library preparation and next-generation sequencing (NGS) techniques, nucleic acids preserved in these samples can now be recovered to yield important genomic information. For this reason, archived FFPE samples have become a rich and precious source of study material. Increasingly, research laboratories are utilizing FFPE samples to profile and understand disease-related genes and mutations.
Multiple factors lead to variability in the quality of the DNA that is isolated from FFPE, including chemical alterations that occur to the nucleic acids during the preservation process, the lack of standardization of the preservation process, and the age of the archived samples. The low quality of the isolated DNA from FFPE samples is expected to generate low-complexity NGS libraries, since the "usable" amount of DNA is low, despite the apparent concentrations of isolated DNA.
In order to create libraries with maximum diversity, selecting the right library preparation method is critical. ThruPLEX technology uses stem-loop adapters that obviate the need for intermediate cleanup steps, maintain a single-tube workflow, and preserve the complexity of the libraries. The final amplification makes use of all of the repaired and ligated molecules, resulting in the high number of unique molecules found in the library (Figure 1). As an added benefit, the time to create libraries is reduced to around two hours. ThruPLEX DNA-seq libraries can also be easily integrated with other applications such as target enrichment. The high diversity of the input material ensures that capture will yield libraries with excellent coverage throughout the targeted regions of the genome.
In this technical note, we demonstrate the performance and features of the ThruPLEX DNA-seq Kit, our general purpose library preparation kit, when used with FFPE-derived DNA. While the recommended input range suggested by the standard protocol for this kit is 0.05–50 ng for non-FFPE samples, here we increased the input amount to 100 ng to compensate for damaged DNA in the FFPE samples. Our results showed that ThruPLEX DNA-seq technology is an excellent choice for preparing NGS libraries from FFPE-derived DNA for Illumina platforms. Compared to other library preparation kits, it provides more diverse libraries while minimizing GC bias. We further demonstrate how the ThruPLEX DNA-Seq Kit can be successfully integrated with Agilent SureSelect Enrichment Kits to carry out targeted sequencing of FFPE samples.