htsintro2013

Transcript

1. Introduction to High-Throughput Sequencing and RNA-seq L Collado-Torres March 6th, 2013 1 / 25
2. High Throughput Sequencing 1 High Throughput Sequencing 2 Sources of variation 2 / 25
3. High Throughput Sequencing DNA1 1Wikipedia. DNA. url: http://en.wikipedia.org/wiki/DNA (visited on 03/05/2013). 3 / 25
4. High Throughput Sequencing Human mRNA2 2Wikipedia. Messanger RNA. url: http://en.wikipedia.org/wiki/Messenger_RNA (visited on 03/05/2013). 4 / 25
5. High Throughput Sequencing Panorama3 3Wendy Weijia Soon, Manoj Hariharan, and Michael P. Snyder. “High-throughput sequencing for biology and medicine”. In: Molecular Systems Biology 9.1 (). url http://www.nature.com/msb/journal/v9/n1/fig_tab/msb201261_F2.html (visited on 03/05/2013). 5 / 25
6. High Throughput Sequencing Prepare DNA4 4Elaine R Mardis. “Next-generation DNA sequencing methods”. In: Annual Review of Genomics and Human Genetics 9 (2008). PMID: 18576944. 6 / 25
7. High Throughput Sequencing Amplify5 5Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010). 7 / 25
8. High Throughput Sequencing PCR6 6SCHOOLWORKHELPER. PCR: Uses, Steps, Purpose. url: http://schoolworkhelper.net/pcr-uses-steps-purpose/ (visited on 03/05/2013). 8 / 25
9. High Throughput Sequencing Sequencing by synthesis7 7Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010). 9 / 25
10. High Throughput Sequencing Analyze cluster images8 8Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010). 10 / 25
11. High Throughput Sequencing HiSeq 20009 9Illumina. HiSeq 2000 Sequencing System. url: http://www.illumina.com/documents/products/datasheets/datasheet_hiseq2000.pdf (visited on 03/05/201 11 / 25
12. High Throughput Sequencing HiSeq 2000 More info on this blog post http://www.politigenomics.com/2010/01/hiseq-2000.html 12 / 25
13. High Throughput Sequencing Other 2nd generation sequencers10 10Lin Liu et al. “Comparison of next-generation sequencing systems”. In: Journal of biomedicine & biotechnology (2012). PMID: 22829749. 13 / 25
14. Sources of variation 1 High Throughput Sequencing 2 Sources of variation 14 / 25
15. Sources of variation Cross-talk11 11Nava Whiteford et al. “Swift: primary data analysis for the Illumina Solexa sequencing platform”. In: Bioinformatics (Oxford, England) 25.17 (2009). PMID: 195496 15 / 25
16. Sources of variation Phasing and pre-phasing12 12Illumina. Pipeline CASAVA User Guide 15003807 ( Pipeline V. 1.4 and Casava V.1.0). 16 / 25
17. Sources of variation Phasing example13 13Nava Whiteford et al. “Swift: primary data analysis for the Illumina Solexa sequencing platform”. In: Bioinformatics (Oxford, England) 25.17 (2009). PMID: 195496 17 / 25
18. Sources of variation Sequence quality14 14Illumina. CASAVA User Guide (15011196 D). url: http://support.illumina.com/downloads/casava_user_guide_15011196.ilmn (visited on 03/05/2013). 18 / 25
19. Sources of variation GC bias15 15Margaret A Taub, Hector Corrada Bravo, and Rafael A Irizarry. “Overcoming bias and systematic errors in next generation sequencing data”. In: Genome Medicin 2.12 (2010). PMID: 21144010. 19 / 25
20. Sources of variation Random primers bias16 16Kasper D Hansen, Steven E Brenner, and Sandrine Dudoit. “Biases in Illumina transcriptome sequencing caused by random hexamer priming”. In: Nucleic Acids Research (2010). PMID: 20395217. 20 / 25
21. Sources of variation Library type17 17Joshua Z Levin et al. “Comprehensive comparative analysis of strand-specific RNA sequencing methods”. In: Nature Methods 7.9 (2010). PMID: 20711195. 21 / 25
22. Sources of variation Batch effects18 18Margaret A Taub, Hector Corrada Bravo, and Rafael A Irizarry. “Overcoming bias and systematic errors in next generation sequencing data”. In: Genome Medicin 2.12 (2010). PMID: 21144010. 22 / 25
23. Sources of variation Biological variability19 19Kasper D Hansen et al. “Sequencing technology does not eliminate biological variability”. In: Nature biotechnology 29.7 (2011). PMID: 21747377. 23 / 25
24. Sources of variation The future Further improvements in library preparation Single cell sequencing Third generation sequencers like Pacific Biosciences And biostatistical methods =) 24 / 25
25. Sources of variation Thanks! Google Calendar https://www.google.com/calendar/embed?src=7hprep991i5prd5l5ftksbsfb8% 40group.calendar.google.com&ctz=America/New_York Slides at http://www.biostat.jhsph.edu/~lcollado/misc/HTSintro.pdf 25 / 25