Evaluation of methods for whole genome and transcriptome sequencing from nanograms of FFPE samples

QC 20161124
The most widely used method for the preservation of clinical tissue specimens is formalin fixation and paraffin embedding (FFPE). Simultaneous analysis of RNA and DNA from samples preserved using this method have long proved problematic, primarily due to lack of material. Here, we describe an attempt to build a complete analysis package for RNA and DNA extracted from single tissue sections. The workflow includes quality control of the extracted material, library preparation and data analysis. We extract DNA with varying integrity from FFPE sections and subject them to whole genome sequencing using two library preparation methods, Illumina TruSeq Nano using the Illumina NeoPrep and Rubicon Genomics ThruPlex. We are able to obtain some usable data, albeit with high duplication rates, demonstrating both the possibilities and challenges of sequencing damaged DNA. Two different approaches to transcriptome sequencing are assessed, the TruSeq RNA Access library preparation kit from Illumina and the SMARTer Stranded Total RNA-Seq Kit - Pico Input from Clonetech. The sequence capture approach of the TruSeq kit is shown to be more robust to low integrity RNA compared to the SMARTer kit. However, the SMARTer kit needs much less starting material and is able to yield data about all transcripts, not just protein coding mRNA.

By the author/editor: Jemt, Anders, 19 ...; Ewels, Philip; Hammarén, Rickar ...; Gruselius, Joel; Borgström, Erik; Wang, Chuan; show more...; Holmberg, Kristi ...; Lundin, Pär; Lundin, Sverker; Gréen, Henrik; Lundeberg, Joaki ...; Käller, Max; show less...

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