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.
आरएनए अनुक्रमण (RNAseq) एक बहुमुखी तरीका है कि पता लगाने और जीन अभिव्यक्ति, म्यूटेशन, जीन fusions, और noncoding RNAs को चिह्नित करने के लिए उपयोग किया जा सकता है। 100 मिलियन अनुक्रमण पढ़ता है और इस तरह के mRNA और noncoding RNAs के रूप में कई आरएनए उत्पादों में शामिल कर सकते हैं – मानक RNAseq 30 की आवश्यकता है। हम कैसे लक्षित RNAseq (कब्जा) एक डेस्कटॉप sequencer का उपयोग कर चुना आरएनए उत्पादों पर ध्यान केंद्रित अध्ययन परमिट प्रदर्शित करता है। RNAseq कब्जा, unannotated कम, या क्षणिक व्यक्त टेप है कि अन्यथा पारंपरिक तरीकों का उपयोग कर RNAseq याद किया जा सकता चिह्नित कर सकते हैं। यहाँ हम सेल लाइनों, ribosomal शाही सेना कमी, सीडीएनए संश्लेषण, बारकोड वाले पुस्तकालयों, संकरण और लक्षित टेप का कब्जा है और एक डेस्कटॉप sequencer पर मल्टीप्लेक्स अनुक्रमण की तैयारी से शाही सेना की निकासी का वर्णन है। हम यह भी कम्प्यूटेशनल विश्लेषण पाइप लाइन है, जो गुणवत्ता नियंत्रण मूल्यांकन, संरेखण, फ्यूजन का पता लगाने, जीन अभिव्यक्ति मात्रा का ठहराव और एकल NUC की पहचान शामिल है, की रूपरेखा तैयारleotide वेरिएंट। इस परख जीन अभिव्यक्ति, जीन fusions, और परिवर्तन को चिह्नित करने के लिए लक्षित प्रतिलेख अनुक्रमण के लिए अनुमति देता है।
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.
RNAseq कैद RNAseq और माइक्रोएरे के बीच मध्यस्थ की रणनीति transcriptome की एक चयनित भाग के मूल्यांकन के लिए दृष्टिकोण है। कैद के फायदे के लिए एक डेस्कटॉप sequencer, उच्च throughput, और जीनोमिक परिवर्तन का पता लगाने पर कम लागत, तेजी से ?…
The authors have nothing to disclose.
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.
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 |