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Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA-Seq Dr. Robert Boissy SWH2048

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Presentation on theme: "Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA-Seq Dr. Robert Boissy SWH2048"— Presentation transcript:

1 Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA-Seq Dr. Robert Boissy SWH2048

2  NGS instruments and data analysis software  RNA-Seq overview  mRNA gene expression  mRNA transcript isoforms  mRNA special cases  miRNA gene expression Outline

3 NGS Instruments and software Glenn TC. (2011) Field guide to next-generation DNA sequencers. Mol Ecol Resour. 11(5):

4 NGS Instruments and software

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7 RNA-Seq overview Li J, Witten DM, Johnstone IM, Tibshirani R. (2011) Normalization, testing, and false discovery rate estimation for RNA-sequencing data. Biostatistics Oct 14.

8 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.

9 RNA-Seq overview Auer PL, Doerge RW. (2010) Statistical design and analysis of RNA sequencing data. Genetics 185:

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

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

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

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

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

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

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

17  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 Oct 14. 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) Fang Z, Cui X. (2011) Design and validation issues in RNA-seq experiments. Brief Bioinform. 12(3):

18 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): Roberts A, Pachter L. (2011) RNA-Seq and find: entering the RNA deep field. Genome Med. 3(11):74. 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):e  Influence of sequencing depth  Influence of mappability

19 mRNA gene expression

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22  Guidelines and reviews mRNA gene expression Standards, Guidelines and Best Practices for RNA-Seq V1.0 (June 2011) The ENCODE Consortium. ndards_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): Garber M, Grabherr MG, Guttman M, Trapnell C. (2011) Computational methods for transcriptome annotation and quantification using RNA-seq. Nat Methods. 8(6): Ramsköld D, Kavak E, Sandberg R. (2012) How to analyze gene expression using RNA-sequencing data. Methods Mol Biol. 802:

23  TopHat and related programs mRNA transcript isoforms Trapnell C, Pachter L, Salzberg SL. (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25(9): Langmead B, Hansen KD, Leek JT. (2010) Cloud-scale RNA-sequencing differential expression analysis with Myrna. Genome Biol. 11(8):R83. Kim D, Salzberg SL. (2011) TopHat-Fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol. 12(8):R72. 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): Roberts A, Pimentel H, Trapnell C, Pachter L. (2011) Identification of novel transcripts in annotated genomes using RNA-Seq. Bioinformatics 27(17): 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.

24 mRNA transcript isoforms

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30  Non-polyadenylated transcripts  Nascent transcripts + co-transcriptional splicing  Circular transcripts  A to I editing mRNA special cases

31 Yang L, Duff MO, Graveley BR, Carmichael GG, Chen LL. (2011) Genomewide characterization of non-polyadenylated RNAs. Genome Biol. 12(2):R16.

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33 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):

34 mRNA special cases

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36 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):e

37 mRNA special cases

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40 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):

41 mRNA special cases

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43  “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, , DOI: / _19 e=1

44  NGS instruments and data analysis software  RNA-Seq overview  mRNA gene expression  mRNA transcript isoforms  mRNA special cases  miRNA gene expression Review


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