Evaluation of Protocols for RRNA Depletion-based RNA Sequencing of Nanogram Inputs of Mammalian Total RNA

Overview

Journal PLoS One

Specialties General Medicine
Science

Date 2019 Nov 1

PMID 31671154

Citations 11

Authors

Simon Haile

Richard D Corbett

Steve Bilobram

Karen Mungall

Bruno M Grande

Heather Kirk

Pawan Pandoh

Tina MacLeod

Helen McDonald

Miruna Bala

Robin J Coope

Richard A Moore

Andrew J Mungall

Yongjun Zhao

Ryan D Morin

Steven J Jones

Marco A Marra

Affiliations

Soon will be listed here.

Abstract

Next generation RNA-sequencing (RNA-seq) is a flexible approach that can be applied to a range of applications including global quantification of transcript expression, the characterization of RNA structure such as splicing patterns and profiling of expressed mutations. Many RNA-seq protocols require up to microgram levels of total RNA input amounts to generate high quality data, and thus remain impractical for the limited starting material amounts typically obtained from rare cell populations, such as those from early developmental stages or from laser micro-dissected clinical samples. Here, we present an assessment of the contemporary ribosomal RNA depletion-based protocols, and identify those that are suitable for inputs as low as 1-10 ng of intact total RNA and 100-500 ng of partially degraded RNA from formalin-fixed paraffin-embedded tissues.

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References

Robertson G, Schein J, Chiu R, Corbett R, Field M, Jackman S . De novo assembly and analysis of RNA-seq data. Nat Methods. 2010; 7(11):909-12. DOI: 10.1038/nmeth.1517. View

Archer S, Shirokikh N, Preiss T . Selective and flexible depletion of problematic sequences from RNA-seq libraries at the cDNA stage. BMC Genomics. 2014; 15:401. PMC: 4045971. DOI: 10.1186/1471-2164-15-401. View

Li P, Conley A, Zhang H, Kim H . Whole-Transcriptome profiling of formalin-fixed, paraffin-embedded renal cell carcinoma by RNA-seq. BMC Genomics. 2014; 15:1087. PMC: 4298956. DOI: 10.1186/1471-2164-15-1087. View

Sturani E, Costantini M, Martegani E, Alberghina L . Level and turnover of polyadenylate-containing ribonucleic acid in Neurospora crassa in different steady states of growth. Eur J Biochem. 1979; 99(1):1-7. DOI: 10.1111/j.1432-1033.1979.tb13224.x. View

Baker S, Bauer S, Beyer R, Brenton J, Bromley B, Burrill J . The External RNA Controls Consortium: a progress report. Nat Methods. 2005; 2(10):731-4. DOI: 10.1038/nmeth1005-731. View

Scolnick J, Dimon M, Wang I, Huelga S, Amorese D . An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples. PLoS One. 2015; 10(7):e0128916. PMC: 4488430. DOI: 10.1371/journal.pone.0128916. View

Iyer M, Chinnaiyan A, Maher C . ChimeraScan: a tool for identifying chimeric transcription in sequencing data. Bioinformatics. 2011; 27(20):2903-4. PMC: 3187648. DOI: 10.1093/bioinformatics/btr467. View

Morlan J, Qu K, Sinicropi D . Selective depletion of rRNA enables whole transcriptome profiling of archival fixed tissue. PLoS One. 2012; 7(8):e42882. PMC: 3416766. DOI: 10.1371/journal.pone.0042882. View

Shanker S, Paulson A, Edenberg H, Peak A, Perera A, Alekseyev Y . Evaluation of commercially available RNA amplification kits for RNA sequencing using very low input amounts of total RNA. J Biomol Tech. 2015; 26(1):4-18. PMC: 4310221. DOI: 10.7171/jbt.15-2601-001. View

10.

Butterfield Y, Kreitzman M, Thiessen N, Corbett R, Li Y, Pang J . JAGuaR: junction alignments to genome for RNA-seq reads. PLoS One. 2014; 9(7):e102398. PMC: 4111418. DOI: 10.1371/journal.pone.0102398. View

11.

Hedegaard J, Thorsen K, Lund M, Hein A, Hamilton-Dutoit S, Vang S . Next-generation sequencing of RNA and DNA isolated from paired fresh-frozen and formalin-fixed paraffin-embedded samples of human cancer and normal tissue. PLoS One. 2014; 9(5):e98187. PMC: 4039489. DOI: 10.1371/journal.pone.0098187. View

12.

Sultan M, Amstislavskiy V, Risch T, Schuette M, Dokel S, Ralser M . Influence of RNA extraction methods and library selection schemes on RNA-seq data. BMC Genomics. 2014; 15:675. PMC: 4148917. DOI: 10.1186/1471-2164-15-675. View

13.

Kuai L, Fang F, Butler J, Sherman F . Polyadenylation of rRNA in Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 2004; 101(23):8581-6. PMC: 423237. DOI: 10.1073/pnas.0402888101. View

14.

Cieslik M, Chugh R, Wu Y, Wu M, Brennan C, Lonigro R . The use of exome capture RNA-seq for highly degraded RNA with application to clinical cancer sequencing. Genome Res. 2015; 25(9):1372-81. PMC: 4561495. DOI: 10.1101/gr.189621.115. View

15.

Svensson V, Natarajan K, Ly L, Miragaia R, Labalette C, Macaulay I . Power analysis of single-cell RNA-sequencing experiments. Nat Methods. 2017; 14(4):381-387. PMC: 5376499. DOI: 10.1038/nmeth.4220. View

16.

Lucas M, Jacobson J, GILES N . Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa. J Bacteriol. 1977; 130(3):1192-8. PMC: 235342. DOI: 10.1128/jb.130.3.1192-1198.1977. View

17.

Grande B, Gerhard D, Jiang A, Griner N, Abramson J, Alexander T . Genome-wide discovery of somatic coding and noncoding mutations in pediatric endemic and sporadic Burkitt lymphoma. Blood. 2019; 133(12):1313-1324. PMC: 6428665. DOI: 10.1182/blood-2018-09-871418. View

18.

Schuierer S, Carbone W, Knehr J, Petitjean V, Fernandez A, Sultan M . A comprehensive assessment of RNA-seq protocols for degraded and low-quantity samples. BMC Genomics. 2017; 18(1):442. PMC: 5460543. DOI: 10.1186/s12864-017-3827-y. View

19.

Adiconis X, Borges-Rivera D, Satija R, DeLuca D, Busby M, Berlin A . Comparative analysis of RNA sequencing methods for degraded or low-input samples. Nat Methods. 2013; 10(7):623-9. PMC: 3821180. DOI: 10.1038/nmeth.2483. View

20.

Sakarya O, Breu H, Radovich M, Chen Y, Wang Y, Barbacioru C . RNA-Seq mapping and detection of gene fusions with a suffix array algorithm. PLoS Comput Biol. 2012; 8(4):e1002464. PMC: 3320572. DOI: 10.1371/journal.pcbi.1002464. View