遺伝子発現およびゲノム変化を特徴づけるために、RNAシーケンシングアッセイをターゲットに
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配列（RNAseq）は、遺伝子発現、突然変異、遺伝子融合、および非コードRNAを検出し、特徴付けるために使用することができる汎用性の高い方法です。億シーケンシング読み取り、そのようなmRNAおよび非コードRNAなどの複数のRNA産物を含めることができます – 標準RNAseq 30が必要です。我々は、ターゲットを絞ったRNAseq（キャプチャ）は、デスクトップ・シーケンサーを使用して、選択したRNA産物にフォーカスされた研究を可能にする方法を示します。 RNAseqキャプチャは、そうでなければ、伝統的なRNAseq方法を使用してミスの可能性があることを、注釈なしの低い、または一時的に発現転写産物を特徴づけることができます。ここでは、細胞株からのRNAの抽出、リボソームRNAの枯渇、cDNA合成、バーコードのライブラリ、ハイブリダイゼーションおよびデスクトップ・シーケンサー上のターゲット転写産物と多重シーケンシングのキャプチャの準備について説明します。我々はまた、品質管理評価、整列、融合検出、遺伝子発現の定量化および単NUCの識別を含むコンピュータ解析パイプラインを、概要leotide変種。このアッセイは、遺伝子発現、遺伝子融合、および変異を特徴付ける標的転写物配列決定を可能にします。
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 mutations4-6.
Capture involves hybridization of complementary oligonucleotides to enrich cDNA libraries for sequencing. The rationale for RNAseq Capture is similar to microarray approaches where complementary oligonucleotides or probes are hybridized to samples and then measured for relative abundance. For microarray technologies, expression is based on relative signal measured for transcripts binding to these probes. Microarrays are thus limited by range, potential background noise from non-specific binding, and cross-hybridization of probes. Furthermore, arrays have limited dynamic range for low and highly expressed transcripts compared to RNAseq1. Microarrays are widely utilized due to their reduced cost and high throughput capacity compared to RNAseq.
Here, we demonstrate a method for RNAseq Capture that offers a middle ground between RNAseq and microarray approaches for evaluating the transcriptome. RNAseq Capture has intermediate throughput, greater dynamic range and sensitivity, and is scaled for fast turnaround on desktop sequencers. RNAseq Capture also requires reduced computational resources in terms of storage space and data processing.
Protocol
Representative Results
Discussion
RNAseq CaptureはRNAseqとマイクロアレイとの間の中間の戦略は、トランスクリプトームの選択した部分を評価するためのアプローチです。キャプチャの利点は、デスクトップ・シーケンサー、高スループット、およびゲノム変化の検出にコスト削減、迅速なターンアラウンドタイムを含みます。この方法は、RNAスプライシングを調べ、単一ヌクレオチド変異体を4-6を検出し 、遺?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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 Foundation, American Lung Association, and Pelotonia.
Materials
| 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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