Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Summary

We describe a targeted RNA sequencing-based method that includes preparation of indexed cDNA libraries, hybridization and capture with custom probes and data analysis to interrogate selected transcripts for gene expression, mutations, and gene fusions. Targeted RNAseq permits cost-effective, rapid evaluation of selected transcripts on a desktop sequencer.

Abstract

RNA sequencing (RNAseq) is a versatile method that can be utilized to detect and characterize gene expression, mutations, gene fusions, and noncoding RNAs. Standard RNAseq requires 30 - 100 million sequencing reads and can include multiple RNA products such as mRNA and noncoding RNAs. We demonstrate how targeted RNAseq (capture) permits a focused study on selected RNA products using a desktop sequencer. RNAseq capture can characterize unannotated, low, or transiently expressed transcripts that may otherwise be missed using traditional RNAseq methods. Here we describe the extraction of RNA from cell lines, ribosomal RNA depletion, cDNA synthesis, preparation of barcoded libraries, hybridization and capture of targeted transcripts and multiplex sequencing on a desktop sequencer. We also outline the computational analysis pipeline, which includes quality control assessment, alignment, fusion detection, gene expression quantification and identification of single nucleotide variants. This assay allows for targeted transcript sequencing to characterize gene expression, gene fusions, and mutations.

Introduction

Whole transcriptome or RNA sequencing (RNAseq) is an unbiased sequencing method to assess all RNA products. The goal of targeted RNAseq (Capture) is a focused evaluation of selected transcripts with increased sensitivity, dynamic range, reduced cost or scale, and increased throughput compared to standard RNAseq. Similar to standard RNAseq, targeted enrichment approaches can be used to evaluate gene expression, multiple RNA species such as mRNA, microRNA (miRNA), lncRNA¹, other noncoding RNAs², gene fusions³, and mutations^4-6.

Capture involves hybridization of complementary oligonucleotides to enri....

Protocol

Note: This protocol describes the simultaneous processing and analysis of four samples. This method is compatible with RNA isolated from cells, fresh frozen tissue and formalin-fixed paraffin-embedded tissue (FFPE). This protocol begins with 50 - 1,000 ng (250 ng recommended) of starting RNA input for each sample.

1. rRNA Depletion and Fragmentation of RNA Procedure

1. rRNA Depletion
   1. Remove elute, prime, fragment mix, rRNA removal mix, rRNA binding buffer and resuspension buffer from -20 °C and thaw at room temperature. Remove elution buffer, rRNA removal beads and RNA/cDNA specific paramagnetic be....

Results

A schematic highlighting key steps in RNAseq Capture is shown in Figure 1. Four cancer cell lines with known mutations were used to demonstrate the effectiveness of the RNAseq Capture technique (K562 with ABL1 fusion, LC2 with RET fusion, EOL1 with PDGFRalpha fusion and RT-4 with FGFR3 fusion). The four samples were pooled together and sequenced with 2x 100 bp reads on a desktop sequencer, which generates FASTQ files. FASTQ files were r.......

Discussion

RNAseq Capture is an intermediate strategy between RNAseq and microarray approaches for evaluating a selected part of the transcriptome. The advantages of Capture include reduced cost, rapid turnaround time on a desktop sequencer, high throughput, and detection of genomic alterations. The method can be adapted to characterize non-coding RNAs²³, detect single nucleotide variants^4-6, examine RNA splicing, and to identify gene fusions or structural rearrangements²⁴. Further, this approach ca.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Thermomixer R	Eppendorf	21516-166
Centrifuge 5417R	Eppendorf	5417R
miRNeasy Mini Kit	Qiagen	217004
Molecular Biology Grade Ethanol	Sigma Aldrich	E7023-6X500ML
Thermoblock 24 x 1.5 ml	Eppendorf	21516-166
MiSeq Reagent Kit v2 (300-cycles)	Illumina	MS-102-2002
MiSeq Desktop Sequencer	Illumina
PhiX Control v3	Illumina	FC-110-3001
TruSeq Stranded Total RNA Kit with RiboZero Gold SetA	Illumina	RS-122-2301
25 rxn xGen® Universal Blocking Oligo - TS-p5	IDT	127040822
25 rxn xGen® Universal Blocking Oligo - TS-p7(6nt)	IDT	127040823
25 rxn xGen® Universal Blocking Oligo - TS-p7(8nt)	IDT	127040824
Agencourt® AMPure® XP - PCR Purification beads	Beckman-Coulter	A63880
Dynabeads® M-270 Streptavidin	Life Technologies	65305
COT Human DNA, Fluorometric Grade, 1 mg	Roche Applied Science	05480647001
Qubit® Assay Tubes	Life Technologies	Q32856
Qubit® dsDNA HS Assay Kit	Life Technologies	Q32851
SeqCap® EZ Hybridization and Wash Kits  (24 or 96 reactions)	Roche NimbleGen	05634261001 or 05634253001
Qubit® 2.0 Fluorometer	Life Technologies	Q32866
10 x 2 ml IDTE pH 8.0 (1x TE Solution)	IDT
Tween20 BioXtra	Sigma	P7949-500ML
Nuclease Free Water	Life Technologies	AM9937
C1000 Touch™ Thermal Cycler with 96–Well Fast Rection Module	Biorad	185-1196
SeqCap EZ Hybridization and Wash Kits	Roche Applied Science	05634253001
SuperScript II Reverse Transcription 200 U/μl	Life Technologies	18064-014
D1000 ScreenTape	Agilent Technol. Inc.	5067-5582
Agencourt RNAClean XP - 40 ml	Beckman Coulter Inc	A63987
RNA ScreenTape	Agilent Technol. Inc.	5067-5576
RNA ScreenTape Ladder	Agilent Technol. Inc.	5067-5578
RNA ScreenTape Sample Buffer	Agilent Technol. Inc.	5067-5577
Sodium Hydroxide	Sigma	72068-100ML
DynaBeads MyOne Streptavidin T1	Life Technologies	65602
DYNAMAG -96 SIDE EACH	Life Technologies	12331D
Chloroform	Sigma	C2432-1L
KAPA HotStart ReadyMix	KAPA Biosystems	KK2602
NanoDrop 2000 Spectrophotometer	Thermo Scientific
My Block Mini Dry Bath	Benchmark	BSH200
D1000 Reagents	Agilent Technol. Inc.	5067- 5583
Vacufuge Plus	Eppendorf	022829861