Targeted Sequencing for Gene Discovery and Quantification Using RNA CaptureSeq

Overview

Journal Nat Protoc

Specialties Biology
Pathology
Science

Date 2014 Apr 8

PMID 24705597

Citations 90

Authors

Tim R Mercer

Michael B Clark

Joanna Crawford

Marion E Brunck

Daniel J Gerhardt

Ryan J Taft

Lars K Nielsen

Marcel E Dinger

John S Mattick

Affiliations

Soon will be listed here.

Abstract

RNA sequencing (RNAseq) samples the majority of expressed genes infrequently, owing to the large size, complex splicing and wide dynamic range of eukaryotic transcriptomes. This results in sparse sequencing coverage that can hinder robust isoform assembly and quantification. RNA capture sequencing (CaptureSeq) addresses this challenge by using oligonucleotide probes to capture selected genes or regions of interest for targeted sequencing. Targeted RNAseq provides enhanced coverage for sensitive gene discovery, robust transcript assembly and accurate gene quantification. Here we describe a detailed protocol for all stages of RNA CaptureSeq, from initial probe design considerations and capture of targeted genes to final assembly and quantification of captured transcripts. Initial probe design and final analysis can take less than 1 d, whereas the central experimental capture stage requires ∼7 d.

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References

Levin J, Yassour M, Adiconis X, Nusbaum C, Thompson D, Friedman N . Comprehensive comparative analysis of strand-specific RNA sequencing methods. Nat Methods. 2010; 7(9):709-15. PMC: 3005310. DOI: 10.1038/nmeth.1491. View

Derveaux S, Vandesompele J, Hellemans J . How to do successful gene expression analysis using real-time PCR. Methods. 2009; 50(4):227-30. DOI: 10.1016/j.ymeth.2009.11.001. View

Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley D . Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc. 2012; 7(3):562-78. PMC: 3334321. DOI: 10.1038/nprot.2012.016. View

Djebali S, Davis C, Merkel A, Dobin A, Lassmann T, Mortazavi A . Landscape of transcription in human cells. Nature. 2012; 489(7414):101-8. PMC: 3684276. DOI: 10.1038/nature11233. View

Dahl F, Gullberg M, Stenberg J, Landegren U, Nilsson M . Multiplex amplification enabled by selective circularization of large sets of genomic DNA fragments. Nucleic Acids Res. 2005; 33(8):e71. PMC: 1087789. DOI: 10.1093/nar/gni070. View

Anders S, McCarthy D, Chen Y, Okoniewski M, Smyth G, Huber W . Count-based differential expression analysis of RNA sequencing data using R and Bioconductor. Nat Protoc. 2013; 8(9):1765-86. DOI: 10.1038/nprot.2013.099. View

Cloonan N, Forrest A, Kolle G, Gardiner B, Faulkner G, Brown M . Stem cell transcriptome profiling via massive-scale mRNA sequencing. Nat Methods. 2008; 5(7):613-9. DOI: 10.1038/nmeth.1223. View

. An integrated encyclopedia of DNA elements in the human genome. Nature. 2012; 489(7414):57-74. PMC: 3439153. DOI: 10.1038/nature11247. View

Mortazavi A, Williams B, McCue K, Schaeffer L, Wold B . Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods. 2008; 5(7):621-8. DOI: 10.1038/nmeth.1226. View

10.

Anders S, Reyes A, Huber W . Detecting differential usage of exons from RNA-seq data. Genome Res. 2012; 22(10):2008-17. PMC: 3460195. DOI: 10.1101/gr.133744.111. View

11.

Quinlan A, Hall I . BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics. 2010; 26(6):841-2. PMC: 2832824. DOI: 10.1093/bioinformatics/btq033. View

12.

Porreca G, Zhang K, Li J, Xie B, Austin D, Vassallo S . Multiplex amplification of large sets of human exons. Nat Methods. 2007; 4(11):931-6. DOI: 10.1038/nmeth1110. View

13.

Turner E, Ng S, Nickerson D, Shendure J . Methods for genomic partitioning. Annu Rev Genomics Hum Genet. 2009; 10:263-84. DOI: 10.1146/annurev-genom-082908-150112. View

14.

DeLuca D, Levin J, Sivachenko A, Fennell T, Nazaire M, Williams C . RNA-SeQC: RNA-seq metrics for quality control and process optimization. Bioinformatics. 2012; 28(11):1530-2. PMC: 3356847. DOI: 10.1093/bioinformatics/bts196. View

15.

Cabili M, Trapnell C, Goff L, Koziol M, Tazon-Vega B, Regev A . Integrative annotation of human large intergenic noncoding RNAs reveals global properties and specific subclasses. Genes Dev. 2011; 25(18):1915-27. PMC: 3185964. DOI: 10.1101/gad.17446611. View

16.

Trapnell C, Hendrickson D, Sauvageau M, Goff L, Rinn J, Pachter L . Differential analysis of gene regulation at transcript resolution with RNA-seq. Nat Biotechnol. 2012; 31(1):46-53. PMC: 3869392. DOI: 10.1038/nbt.2450. View

17.

Trapnell C, Williams B, Pertea G, Mortazavi A, Kwan G, van Baren M . Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 2010; 28(5):511-5. PMC: 3146043. DOI: 10.1038/nbt.1621. View

18.

Clark M, Chen R, Lam H, Karczewski K, Chen R, Euskirchen G . Performance comparison of exome DNA sequencing technologies. Nat Biotechnol. 2011; 29(10):908-14. PMC: 4127531. DOI: 10.1038/nbt.1975. View

19.

Mezlini A, Smith E, Fiume M, Buske O, Savich G, Shah S . iReckon: simultaneous isoform discovery and abundance estimation from RNA-seq data. Genome Res. 2012; 23(3):519-29. PMC: 3589540. DOI: 10.1101/gr.142232.112. View

20.

Harrow J, Denoeud F, Frankish A, Reymond A, Chen C, Chrast J . GENCODE: producing a reference annotation for ENCODE. Genome Biol. 2006; 7 Suppl 1:S4.1-9. PMC: 1810553. DOI: 10.1186/gb-2006-7-s1-s4. View