Ciblées ARN Séquençage Assay pour caractériser l'expression génique et génomique Transformations
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
séquençage de l'ARN (RNA-seq) est une méthode polyvalente qui peut être utilisée pour détecter et caractériser l'expression des gènes, des mutations, des fusions de gènes, et ARNs non codants. Norme exige RNA-seq 30-100000000 séquençage lit et peut inclure plusieurs produits d'ARN tels que l'ARNm et ARN non codantes. Nous démontrons comment ciblée RNA-seq (capture) permet une étude ciblée sur les produits d'ARN sélectionnés à l'aide d'un séquenceur de bureau. RNA-seq capture peut caractériser les transcriptions annotées, faibles, ou exprimés de façon transitoire qui pourraient autrement être manquées en utilisant des méthodes traditionnelles RNA-seq. Nous décrivons ici l'extraction d'ARN à partir de lignées cellulaires, l'ARN ribosomique, l'épuisement de la synthèse d'ADNc, la préparation de bibliothèques de codes à barres, l'hybridation et la capture des produits de transcription cibles, et le séquençage multiplex sur un séquenceur de bureau. Nous présentons également le pipeline d'analyse informatique, qui comprend l'évaluation du contrôle de la qualité, l'alignement, la détection de la fusion, l'expression du gène de quantification et d'identification de simple nucdes variants de leotide. Ce dosage permet un séquençage de transcription ciblé pour caractériser l'expression des gènes, des fusions de gènes et des 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 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
RNA-seq Capture est une stratégie intermédiaire entre RNA-seq et microarray approches pour l'évaluation d'une partie sélectionnée du transcriptome. Les avantages de la capture comprennent le coût, le délai d'exécution rapide réduits sur un séquenceur de bureau, à haut débit, et la détection des altérations génomiques. Le procédé peut être adapté pour caractériser ARN non-codants 23, détecter seul nucléotide variantes 4-6, examiner épissage de l' ARN, et d'…
Declarações
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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