Introduction to Next-Generation Sequencing (NGS)

Analysis of Transcription using RNA-Seq

Dr. Robert BoissySWH2048

rboissy@unmc.edu

NGS instruments and data analysis software

RNA-Seq overview

mRNA gene expression

mRNA transcript isoforms

mRNA special cases

miRNA gene expression

Outline

NGS Instruments and software

http://seqanswers.com/

Glenn TC. (2011) Field guide to next-generation DNA sequencers.Mol Ecol Resour. 11(5):759-69.

http://www.ncbi.nlm.nih.gov/pubmed/21592312http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0998.2011.03024.x/abstracthttp://www.molecularecologist.com/next-gen-fieldguide/

NGS Instruments and software

NGS Instruments and software

NGS Instruments and software

RNA-Seq overview

Li J, Witten DM, Johnstone IM, Tibshirani R. (2011) Normalization, testing, and false discovery rate estimation for RNA-sequencing data. Biostatistics. 2011 Oct 14.http://www.ncbi.nlm.nih.gov/pubmed/22003245

McIntyre LM, Lopiano KK, Morse AM, Amin V, Oberg AL, Young LJ, Nuzhdin SV. (2011) RNA-seq: technical variability and sampling. BMC Genomics. 12:293.http://www.ncbi.nlm.nih.gov/pubmed/21645359

RNA-Seq overview

Auer PL, Doerge RW. (2010) Statistical design and analysis of RNA sequencing data. Genetics 185:405-16. http://www.ncbi.nlm.nih.gov/pubmed?term=20439781

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 333

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 338

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 338

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 338

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 350

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 353

RNA-Seq overview

Pevsner, J (2009) Bioinformatics and Functional Genomics Wiley-Blackwell p. 354

Biostatistical expertise is essential

Study design and power estimates need to be worked out before sequencing

mRNA gene expression

Li J, Witten DM, Johnstone IM, Tibshirani R. (2011) Normalization, testing, and false discovery rate estimation for RNA-sequencing data. Biostatistics. 2011 Oct 14.http://www.ncbi.nlm.nih.gov/pubmed/22003245

McCarthy DJ, Chen Y, Smyth GK. (2012) Differential expression analysis of multifactor RNA-Seq experiments with respect to biological variation. Nucleic Acids Res. (Feb. 6) http://www.ncbi.nlm.nih.gov/pubmed/22287627

Fang Z, Cui X. (2011) Design and validation issues in RNA-seq experiments. Brief Bioinform. 12(3):280-7. http://www.ncbi.nlm.nih.gov/pubmed/21498551

mRNA gene expression

Mercer TR, Gerhardt DJ, Dinger ME, Crawford J, Trapnell C, Jeddeloh JA, Mattick JS, Rinn JL. (2011) Targeted RNA sequencing reveals the deep complexity of the human transcriptome. Nat Biotechnol. 30(1):99-104. http://www.ncbi.nlm.nih.gov/pubmed/22081020

Roberts A, Pachter L. (2011) RNA-Seq and find: entering the RNA deep field. Genome Med. 3(11):74. http://www.ncbi.nlm.nih.gov/pubmed/22113004

Derrien T, Estellé J, Marco Sola S, Knowles DG, Raineri E, Guigó R, Ribeca P. (2012) Fast computation and applications of genome mappability. PLoS One. 7(1):e30377. http://www.ncbi.nlm.nih.gov/pubmed/22276185

Influence of sequencing depth

Influence of mappability

mRNA gene expression

mRNA gene expression

Guidelines and reviews

mRNA gene expression

Standards, Guidelines and Best Practices for RNA-Seq V1.0 (June 2011) The ENCODE Consortium. http://encodeproject.org/ENCODE/protocols/dataStandards/ENCODE_RNAseq_Standards_V1.0.pdf

Jiang L, Schlesinger F, Davis CA, Zhang Y, Li R, Salit M, Gingeras TR, Oliver B. (2011) Synthetic spike-in standards for RNA-seq experiments. Genome Res. 21(9):1543-51. http://www.ncbi.nlm.nih.gov/pubmed/21816910

Garber M, Grabherr MG, Guttman M, Trapnell C. (2011) Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 8(6):469-77. http://www.ncbi.nlm.nih.gov/pubmed/21623353

Ramsköld D, Kavak E, Sandberg R. (2012) How to analyze gene expression using RNA-sequencing data. Methods Mol Biol. 802:259-74. http://www.ncbi.nlm.nih.gov/pubmed/22130886

TopHat and related programs

mRNA transcript isoforms

Trapnell C, Pachter L, Salzberg SL. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9):1105-11.http://www.ncbi.nlm.nih.gov/pubmed/19289445 http://tophat.cbcb.umd.edu/

Langmead B, Hansen KD, Leek JT. (2010) Cloud-scale RNA-sequencing differential expression analysis with Myrna. Genome Biol. 11(8):R83. http://www.ncbi.nlm.nih.gov/pubmed/20701754 http://bowtie-bio.sourceforge.net/myrna/index.shtml

Kim D, Salzberg SL. (2011) TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol. 12(8):R72.http://www.ncbi.nlm.nih.gov/pubmed/21835007 http://tophat-fusion.sourceforge.net/

Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, Salzberg SL, Wold BJ, Pachter L. (2010) Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol. 28(5):511-5. http://www.ncbi.nlm.nih.gov/pubmed/20436464 http://cufflinks.cbcb.umd.edu/

Roberts A, Pimentel H, Trapnell C, Pachter L. (2011) Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27(17):2325-9. http://www.ncbi.nlm.nih.gov/pubmed/21697122

Roberts A, Trapnell C, Donaghey J, Rinn JL, Pachter L. (2011) Improving RNA-Seq expression estimates by correcting for fragment bias. Genome Biol. 12(3):R22. http://www.ncbi.nlm.nih.gov/pubmed/21410973

mRNA transcript isoforms

mRNA transcript isoforms

Non-polyadenylated transcripts

Nascent transcripts + co-transcriptional splicing

Circular transcripts

A to I editing

mRNA special cases

Yang L, Duff MO, Graveley BR, Carmichael GG, Chen LL. (2011) Genomewide characterization of non-polyadenylated RNAs. Genome Biol. 12(2):R16. http://www.ncbi.nlm.nih.gov/pubmed/21324177

Ameur A, Zaghlool A, Halvardson J, Wetterbom A, Gyllensten U, Cavelier L, Feuk L. (2011) Total RNA sequencing reveals nascent transcription and widespread co-transcriptional splicing in the human brain. Nat Struct Mol Biol. 18(12):1435-40. http://www.ncbi.nlm.nih.gov/pubmed/22056773

mRNA special cases

mRNA special cases

Salzman J, Gawad C, Wang PL, Lacayo N, Brown PO. (2012) Circular RNAs Are the Predominant Transcript Isoform from Hundreds of Human Genes in Diverse Cell Types. PLoS One. 7(2):e30733. http://www.ncbi.nlm.nih.gov/pubmed/22319583

mRNA special cases

mRNA special cases

mRNA special cases

Bahn JH, Lee JH, Li G, Greer C, Peng G, Xiao X. (2011) Accurate identification of A-to-I RNA editing in human by transcriptome sequencing. Genome Res. 22(1):142-50. http://www.ncbi.nlm.nih.gov/pubmed/21960545

mRNA special cases

“Small RNA-Seq” is also very important

miRNA gene expression

For a recent review see:

Preethi H. Gunaratne, Cristian Coarfa, Benjamin Soibam and Arpit Tandon (2012) miRNA Data Analysis: Next-Gen Sequencing. In: Next-generation MicroRNA expression profiling technology, Fan, J.B. (Ed.)Methods in Molecular Biology, Vol. 822, 273-288, DOI: 10.1007/978-1-61779-427-8_19http://www.springerlink.com/content/p21116x3572581r7/#section=999898&page=1

NGS instruments and data analysis software

RNA-Seq overview

mRNA gene expression

mRNA transcript isoforms

mRNA special cases

miRNA gene expression

Review