Abstract 3644: Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification

Abstract

Abstract Massively parallel sequencing (MPS) is increasingly used in the laboratory and clinic to identify genomic factors contributing to tumorigenesis, such as somatic mutations, DNA insertions and deletions, transcriptome and epigenetic changes, and chromosomal abnormalities. Because tumor specimens are often quite limited or are from rare and precious samples, it is necessary to prepare sequencing libraries from minimal amounts of DNA. Indeed, it can be challenging to extract sufficient amounts of starting material from fresh-frozen paraffin embedded (FFPE) samples for library construction, and frequently samples are not processed because they do not reach minimum requirements. Therefore, it is of great importance to improve currently available protocols to process low yield FFPE-derived DNA for MPS. This will allow for the inclusion of more tumor samples to be sequenced per cohort, resulting in increased ability to identify genomic factors involved in tumorigenesis. Here, we optimized the KAPA Library Preparation Kit (Kapa Biosystems, Wilmington, MA) to successfully perform library construction from low-yield FFPE and fresh frozen samples. The kit normally requires an input of at least 100 ng of DNA. However, we were able to modify the protocol to prepare libraries from as little as 10 ng of starting material by optimizing the ratio of adapter:input DNA and SPRI-clean-up, performing low-volume reactions, and using IDT universal blockers during hybrid capture. Additional library enrichment PCR was not required. Library yields were sufficient for downstream hybrid capture and sequencing, and sequencing metrics were comparable to samples that were prepped using the manufacturer's recommendations. Experiments are underway to demonstrate that library complexity remains unchanged. Citation Format: Aaron R. Thorner, Liuda Ziaugra, Matthew D. Ducar, Ling Lin, Angelica Laing, Haley A. Coleman, Suzanne R. McShane, Andrea Clapp, Rachel R. Paquette, Bruce M. Wollison, Johann Hoeftberger, Neil A. Patel, Samuel S. Hunter, Monica D. Manam, Laura E. MacConaill, William C. Hahn, Matthew L. Meyerson, Paul van Hummelen. Optimization of library construction for massively parallel sequencing using low-input, FFPE-derived DNA without additional PCR amplification. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3644.