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Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Biology

Targeted RNA Sequencing Assay to Characterize Gene Expression and Genomic Alterations

Published: August 4th, 2016

DOI:

10.3791/54090

1Department of Internal Medicine, Division of Medical Oncology, Comprehensive Cancer Center, The Ohio State University, 2Department of Pharmacology, The Ohio State University

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.

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.

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 mutations4-6.

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

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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 .......

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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.......

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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 variants4-6, examine RNA splicing, and to identify gene fusions or structural rearrangements24. Further, this approach ca.......

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We give special thanks to Ezra Lyon, Eliot Zhu, Michele Wing, Esko Kautto and Eric Samorodnitsky for technical support. We would also like to thank Jenny Badillo for her administrative support for our team. We acknowledge the Ohio Supercomputer Center (OSC) for providing disk space, processing capacity, and support to run our analyses. We thank the Comprehensive Cancer Center (CCC) at The Ohio State University Wexner Medical Center for their administrative support of this work. S.R. and Team are supported by the American Cancer Society (MRSG-12-194-01-TBG), a Prostate Cancer Foundation Young Investigator Award, NHGRI (UM1HG006508-01A1), Fore Cancer Research Foundatio....

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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 1.5ml 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, 1mg 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 200U/ul Life Technologies 18064-014
D1000 ScreenTape Agilent Technol. Inc. 5067-5582
Agencourt RNAClean XP -40ml 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 

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