htsintro2013

Transcript

1. Introduction to High-Throughput Sequencing and RNA-seq
   L Collado-Torres
   March 6th, 2013
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2. High Throughput Sequencing
   1 High Throughput Sequencing
   2 Sources of variation
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3. High Throughput Sequencing
   DNA1
   1Wikipedia. DNA. url: http://en.wikipedia.org/wiki/DNA (visited on 03/05/2013).
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4. High Throughput Sequencing
   Human mRNA2
   2Wikipedia. Messanger RNA. url: http://en.wikipedia.org/wiki/Messenger_RNA (visited on 03/05/2013).
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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).
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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.
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7. High Throughput Sequencing
   Amplify5
   5Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010).
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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
   

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9. High Throughput Sequencing
   Sequencing by synthesis7
   7Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010). 9 / 25
   

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10. High Throughput Sequencing
    Analyze cluster images8
    8Michael L. Metzker. “Sequencing technologies — the next generation”. In: Nat Rev Genet 11.1 (2010).
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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
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12. High Throughput Sequencing
    HiSeq 2000
    More info on this blog post http://www.politigenomics.com/2010/01/hiseq-2000.html
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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.
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14. Sources of variation
    1 High Throughput Sequencing
    2 Sources of variation
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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
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16. Sources of variation
    Phasing and pre-phasing12
    12Illumina. Pipeline CASAVA User Guide 15003807 ( Pipeline V. 1.4 and Casava V.1.0).
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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
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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).
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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.
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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
    

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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.
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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
    

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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.
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24. Sources of variation
    The future
    Further improvements in library preparation
    Single cell sequencing
    Third generation sequencers like Pacific Biosciences
    And biostatistical methods =)
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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
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