Targeted RNA–Seq

Targeted RNA sequencing (RNA-seq) is a highly accurate method for selecting and sequencing specific transcripts. It isolates and analyzes only genes of your interest, allowing for higher sequencing depth and higher throughput. Targeted RNA-seq can be achieved by enrichment or amplicon-based methods, which are ideal for expression analysis of FFPE tumor specimens and can identify mutations, splicing mutations and fusions.

Overview

Targeted RNA-seq is a next-generation sequencing (NGS)-based gene expression profiling method that provides quantitative and qualitative information of selective genes. For enrichment-based method, the process of targeted RNA-seq includes cDNA synthesis, NGS library preparation, hybridization, capture and sequencing. The aim is to evaluate selected transcripts through increasing sensitivity and dynamic range, hence reducing cost or scale and increasing depth. Similar to standard RNA-seq, targeted RNA-seq can be used to evaluate gene expression, detect multiple RNA species such as mRNA, microRNA (miRNA), lncRNA, and other non-coding RNAs, and discover mutations and gene fusions. This approach improves sequencing coverage by targeting and capturing hundreds of genes within a single run, enabling the sensitive detection of rare or low-expressed transcripts.

Targeted RNA-seq has a higher specificity and improves the ability of mutation detection, enabling the discovery of novel chimeric transcripts and providing the complete gene expression of targeted RNA molecules. It allows researchers to monitor changes in gene expression, identify fusion genes and monitor genetic variants. The precise capture and deep sequencing abilities of targeted RNA-seq provide a deeper understanding of the biology of fusion gene biology, RNA expression, and RNA variants.

Project Workflow

Bioinformatics Analysis Pipeline

In-depth data analysis:

• Differential expression analysis of transcripts
• Detect novel and rare transcript variants
• GO and KEGG annotation analysis
• Alternative splicing analysis
• SNPs/InDels analysis
• Fusion calling

Sample Requirements

• RNA sample (concentration ≥ 20 ng/uL, quantity ≥ 2 ug)
• 1.8 ≤ A260/A280 ≤ 2.0, RIN ≥ 7.0.
• Please make sure that the RNA is not degraded.

Sample storage: RNA can be dissolved in ethanol or RNA-free ultra-pure water and stored at -80°C. RNA should avoid repeated freezing and thawing.

Deliverable: FastQ, BAM, coverage summary, QC report, custom bioinformatics analysis.

References:

1. Erin E H, Ira W D, Danson W, et al. Diagnosis of fusion genes using targeted RNA sequencing. Nature communications, 2019, 5, 10(1):1388.
2. Mercer T R, Gerhardt D J, Dinger M E, et al. Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol, 2011, 13, 30(1): 99-104.
3. Fu G K, Xu W, Wilhelmy J, et al. Molecular indexing enables quantitative targeted RNA sequencing and reveals poor efficiencies in standard library preparations. Proc Natl Acad Sci U S A, 2014, 111(5): 1891-1896.