introduction to next-generation sequencing (ngs) analysis of transcription using rna-seq dr. robert...
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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
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