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Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA- Seq

Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA- Seq Dr. Robert Boissy SWH2048 rboissy@unmc.edu. NGS instruments and data analysis software RNA- Seq overview mRNA gene expression mRNA transcript isoforms mRNA special cases miRNA gene expression.

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Introduction to Next-Generation Sequencing (NGS) Analysis of Transcription using RNA- Seq

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

  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 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/21592312 http://onlinelibrary.wiley.com/doi/10.1111/j.1755-0998.2011.03024.x/abstract http://www.molecularecologist.com/next-gen-fieldguide/

  4. NGS Instruments and software

  5. NGS Instruments and software

  6. NGS Instruments and software

  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. 2011 Oct 14. http://www.ncbi.nlm.nih.gov/pubmed/22003245

  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. http://www.ncbi.nlm.nih.gov/pubmed/21645359

  9. 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

  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. 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

  18. mRNA gene expression • Influence of sequencing depth • Influence of mappability 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

  19. mRNA gene expression

  20. mRNA gene expression

  21. 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

  22. 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

  23. mRNA transcript isoforms

  24. mRNA transcript isoforms

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

  26. 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

  27. 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 StructMol Biol. 18(12):1435-40. http://www.ncbi.nlm.nih.gov/pubmed/22056773

  28. mRNA special cases

  29. 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

  30. mRNA special cases

  31. mRNA special cases

  32. 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

  33. mRNA special cases

  34. “Small RNA-Seq” is also very important miRNA gene expression For a recent review see: PreethiH. Gunaratne, CristianCoarfa, Benjamin Soibam and ArpitTandon (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_19 http://www.springerlink.com/content/p21116x3572581r7/#section=999898&page=1

  35. 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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