RNA-seq for biomarker identification using the xGen ™ Broad-Range RNA Library Prep Kit and xGen Custom Hyb Panels A capable workflow for transcriptome sequencing and targeted RNA-seq with FFPE samples

Biomarker research provides potential targets that can indicate a normal biological process, a pathogenic process, or whether a drug is effective in its mode of action. An efficient workflow for biomarker research and discovery from formalin-fixed, paraffin-embedded (FFPE) samples is presented here. This workflow includes library preparation from isolated FFPE RNA using the xGen Broad-Range RNA Library Prep Kit , target enrichment using an xGen Custom Hyb Panel, followed by sequencing on an Illumina ® platform. The workflow shows consistency across technical replicates and multiple mass inputs. The transcript-specific target enrichment capture panel design bypasses the need for rRNA depletion and produces deeper target coverage, thereby improving identification of low-expressed and rare fusion transcripts over whole transcriptome sequencing with FFPE RNA. Using highly characterized ALK-RET-ROS1 reference standards, this application note shows the ability to capture and call known gene fusions and presents a new hybridization capture panel design approach for identifying novel gene fusions to support oncology research at the biomarker discovery phase. By using a targeted RNA-seq approach, gene fusions were identified at higher rates than transcriptome libraries while using 10-fold less sequencing reads—drastically saving on sequencing costs. (ddPCR)-confirmed EML4-ALK, CCDC6-RET and SLC34A2-ROS1 gene fusions, using two methods. The first workflow relied on ribosomal RNA depletion prior to generation of whole transcriptome libraries. The second workflow used hybridization capture of total RNA libraries to enrich for specific regions of the transcriptome using one of two xGen Custom Hybridization Capture Panels targeting a subset of non-small cell lung cancer (NSCLC)-related genes and gene fusions. Whole transcriptome and targeted sequencing results demonstrate a high-performing workflow with consistency between technical replicates and highly correlated gene expression information. While some gene fusions were not identified in the whole transcriptome libraries, all hybridization captured libraries allowed for the identification of all targeted gene fusions, at significantly higher rates than transcriptome libraries, and while using 10-fold less sequencing reads than transcriptome sequencing. The ALK-RET-ROS1 targeted FFPE RNA Fusion Reference Standard is a highly characterized control material used to assess fusion identification for a variety of assays, including RNA-seq. FFPE RNA (Horizon HD784) was extracted using the Qiagen RNeasy ™ FFPE Kit with deparaffinization solution according to the manufacturer’s protocol. RIN scores of 3.0–3.3 and DV 200 scores (>70%) were determined using an Agilent 2100 bioanalyzer. 10 ng or 50 ng of extracted total RNA was rRNA depleted in triplicate using the Lexogen RiboCop ™ rRNA Depletion Kit HMR V2. Transcriptome libraries were generated from all rRNA-depleted RNA ( n = 6) following the xGen Broad-Range RNA Library Prep Kit protocol and using modifications outlined in Appendix C: Adjustments for xGen Hybridization Capture Panels. Libraries were sequenced using a NextSeq ™ 550 (Illumina) using 2 x 150 paired-end reads and subsampled to 40 million reads per sample. For analysis, 10 bases were trimmed from the beginning of each read due to the low complexity tail added during the Adaptase step (for more information, see the technical note Tail trimming for better data ).