Sarah Trusiak1, Michelle Dubuke1, Bhavin Patel2, and Emily I. Chen1
Purpose: This multi-omics sample preparation workflow enables extraction of DNA, RNA and peptides from multiple sample types, including cells, fresh frozen tissue and FFPE. The isolated DNA and RNA are fully compatible with Next-Generation Sequencing (NGS) and the peptides are fully compatible with Mass Spectrometry (mass spec).
Methods: The multi-omics extraction protocol combines the Thermo Scientific™ EasyPep™ Mini MS Sample Prep Kit and the Applied Biosystems™ MagMAX™ FFPE DNA/RNA Ultra Kit with modified protocols to allow for successive isolation of peptides, DNA, and RNA from the same sample. DNA and RNA compatibility with NGS was verified on the Ion Torrent™ Ion™ Chef and S5 prime system. Peptide compatibility with mass spec was verified on the Thermo Scientific™ Orbitrap Fusion™ Lumos™ Tribrid™ mass spectrometer.
Results: In this study we applied the multi-omics sample prep workflow to three applications; targeted signaling pathway and transcriptomic analysis in human cells, molecular profiling in human fresh frozen tissue, and molecular profiling with DNA mutation analysis in human FFPE cancer tissue. For human cells, the multi-omics extraction method yielded high quality DNA, RNA and peptides, with RNA and peptide yields similar to or better than standard extraction methods. We observed very similar comparative expression counts for 13 AKT/mTOR pathway genes for cells with and without IGF treatment from RNA extracted by the multi-omics verses standard methods. A much larger transcriptomic analysis showed an excellent correlation between RNA from human cells extracted by either method. When applying the multi-omics samples preparation workflow to human fresh frozen tissue, there was no loss in DNA and RNA quality compared to standard extraction methods. NGS results from an Oncomine panel targeting >3700 amplicons were nearly identical for DNA from the two extraction methods, with target detection above 95% and uniformity of amplicon coverage above 98%. Multi-omics extracted peptides from the fresh frozen tissue were fully compatible with mass spec and had 70% of the protein group IDs of the standard extracted peptides. Peptides isolated by the multi-omics protocol had no bias for any particular cellular compartment. Finally, the multi-omics sample preparation protocol could be successfully applied to human breast FFPE tissues and yielded plenty of DNA, RNA and peptides for downstream analysis. We again observed high quality NGS DNA sequencing results using a comprehensive Oncomine panel on the FFPE multi-omics extracted DNA, with target detection above 95% and uniformity of amplicon coverage above 96%. Further, we were able to detect five clinically relevant DNA mutations in the breast cancer samples that had been orthogonally confirmed by SNP qPCR.
1Thermo Fisher Scientific Precision Medicine Science Center, Cambridge, MA 02139; 2ThermoFisher Scientific, Rockford, IL 61102