RNA 시퀀싱 분석은 유전자 발현 및 게놈 변경을 특성화를 대상으로
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 서열 (RNAseq)는 유전자 발현 돌연변이 유전자 융합체, 및 비 암호화 RNA를 검출하고 특성화하는 데에 이용 될 수있는 다목적 방법이다. 1 억 순서 읽고 등의 mRNA 및 비 암호화 RNA를 여러 RNA 제품을 포함 할 수있다 – 표준 RNAseq (30)가 필요합니다. 우리는 타겟 RNAseq (캡처) 바탕 화면 시퀀서를 사용하여 선택 RNA 제품에 초점을 맞춘 연구를 허용하는 방법을 보여줍니다. RNAseq 캡처 그렇지 않으면 기존의 RNAseq 방법을 사용하여 놓칠 수있다, 주석이 달려 있지 않은 저, 또는 일시적으로 표현 된 성적 증명서를 특성화 할 수 있습니다. 여기에서 우리는 세포 라인, 리보솜 RNA 고갈, cDNA 합성, 바코드 라이브러리, 바탕 화면 시퀀서에 하이브리드 및 목표 성적 증명서의 캡처 및 다중 서열의 준비에서 RNA의 추출을 설명합니다. 또한 품질 관리 평가, 정렬 검출 융합 유전자 발현의 정량화 및 단일 NUC의 식별을 포함하는 연산 파이프 라인을 분석, 개요leotide 변형. 이 분석은 유전자 발현, 유전자 융합, 돌연변이의 특성을 타겟팅 사체 서열을 허용한다.
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 캡처 RNAseq 및 마이크로 어레이 사이의 중간 전략은 사체의 선택된 부분을 평가하기위한 접근이다. 캡처의 이점은 유전자 변형의 데스크탑 시퀀서, 높은 처리량, 및 검출에 선정 신속한 처리 시간 감소를 포함한다. 이 방법은, 비 코딩 RNA를 (23)의 특성을 단일 염기를 검출하도록 구성 될 수있는 RNA의 스 플라이 싱을 조사하고 유전자 융합체 또는 구조적 재 배열 (24)을 식별하기…
Divulgations
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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