An Introduction to Second-­Generation Sequencing: Mapping and Counting

932f0888aeedcd7600da2e02fe49cd7e?s=47 Sean Davis
January 12, 2012

An Introduction to Second-­Generation Sequencing: Mapping and Counting

Second-generation sequencing technologies are changing genomics research and many groups want to get in on the action. This talk simply outlines some of the dos and don'ts that I pass along to folks before they embark on a sequencing project. Included are a few slides on mRNA, miRNA, genomic tumor/normal, structural variation, and DNAse/ChIP sequencing.

932f0888aeedcd7600da2e02fe49cd7e?s=128

Sean Davis

January 12, 2012
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  1. 1.

    Sean  Davis,  M.D.,  Ph.D. Gene0cs  Branch,  Center  for  Cancer  Research

    Na0onal  Cancer  Ins0tute Na0onal  Ins0tutes  of  Health h>p://watson.nci.nih.gov/~sdavis An  Introduc+on  to   Second-­‐Genera+on  Sequencing: Mapping  and  Coun+ng 1 Friday, January 13, 2012
  2. 4.

    phenotype Gene  Copy   Number Sequence   Varia4on Chroma4n  

    Structure  and   Func4on Gene  Expression Transcrip4onal   Regula4on DNA  Methyla4on 4 Friday, January 13, 2012
  3. 5.

    phenotype Gene  Copy   Number Sequence   Varia4on Chroma4n  

    Structure  and   Func4on Gene  Expression Transcrip4onal   Regula4on DNA  Methyla4on Pa+ent  and   Popula+on   Characteris+cs 4 Friday, January 13, 2012
  4. 6.

    5 Sequencing  Instruments • Illumina – GAIIx,  MiSeq,  HiSeq  2000,

     HiSeq  2500 • Life  Technologies – SoLID,  Ion  Torrent,  Proton • PacBio – PacBio  RS • Roche – 454  GS  Junior,  GS  FLX • Complete  Genomics* 5 Friday, January 13, 2012
  5. 23.

    DNAse-­‐chip  Method Crawford,  G.E.,  Davis,  S.,  Scacheri,  P.C.,  Renaud,  G.,

     Halawi,  M.J.,  Erdos,  M.R.,  Green,  R.,   Meltzer,  P.S.,  Wolfsberg,  T.G.,  and  Collins,  F.S.  Nat  Methods,  2006 22 Friday, January 13, 2012
  6. 25.

    DNAse  HS  Sites  and  Gene  Expression  DNAse  HS  sites

     near   transcrip0on  start  sites   are  associated  with   ac0vely  transcribed   genes. 24 Friday, January 13, 2012
  7. 26.

    Barski  A,  Zhao  K.  Genomic  loca0on  analysis  by  ChIP-­‐Seq.  J

     Cell  Biochem.  2009 25 Friday, January 13, 2012
  8. 27.

    26 Tag  Count  Sequencing  Pointers • Good  an0body  necessary  for

     good  results • Posi0ve  controls  for  quality  control • Experimental  controls  and  experimental  design   are  important • Need  replicates  if  asking  about  generalizability • Amount  of  sequencing  varies  from  one   applica0on  to  the  next • Mul0plexing  and  indexing 26 Friday, January 13, 2012
  9. 33.

    RNA-­‐Seq  Applica0ons • Transcript  quan0fica0on  and  differen0al   expression •

    Splicing  and  isoform  quan0fica0on • Fusion  gene  discovery • Novel  transcript  discovery • Allele-­‐specific  expression • Variant  discovery  and  quan0fica0on • RNA-­‐edi0ng  observa0on 32 32 Friday, January 13, 2012
  10. 36.

    35 RNA-­‐Seq  Pointers • Replicates  necessary  for  any  sta0s0cal  inference

    – Not  doing  replicates  has  a  cost! • Easier  to  find  differen0al  expression  for  longer  and   more  highly  expressed  genes – Downstream  Gene  Set  Analysis  (GO,  Pathways)  need  to   be  adjusted • Quality  of  transcriptome  annota0on  important • Mapping  and  quan0fica0on  is  complicated • Microarrays  are  not  ex0nct  for  transcriptome   profiling 35 Friday, January 13, 2012
  11. 39.

     Long  oligos  synthesized  on   arrays  (DNA)  RNA

     baits  synthesized  from   DNA  oligo  template  RNA  baits  hybridized  to   DNA  sequencing  library  Targets  captured  using   beads  and  bio0n-­‐labeled   baits  RNA  bait  degraded,  leaving   sequencing  library  enriched   for  target  regions 38 Friday, January 13, 2012
  12. 44.

    The  Cancer  Genome  Atlas  Research  Network  Nature  000,  1-­‐8  (2008)

     doi:10.1038/nature07385 Frequent  gene0c  altera0ons  in  three  cri0cal  signalling  pathways. 43 Friday, January 13, 2012
  13. 48.
  14. 49.
  15. 60.

    Public  Data  1000  Genomes,  HapMap,  Encyclopedia  of  DNA  

    Elements  (ENCODE),  The  Cancer  Genome  Atlas   (TCGA),  COSMIC  database  NCBI  Gene  Expression  Omnibus  (GEO)  Sequence  Read  Archive  (SRA)  Hundreds  of  datasets  not  submi>ed  publicly  or   housed  only  in  proprietary  databases 59 Friday, January 13, 2012
  16. 61.

    Phenotype Gene  Copy   Number Sequence   Varia4on Chroma4n  

    Modifica4on Gene   Expression Transcrip4onal   Regula4on DNA   Methyla4on 60 Friday, January 13, 2012
  17. 62.

    Phenotype Gene  Copy   Number Sequence   Varia4on Chroma4n  

    Modifica4on Gene   Expression Transcrip4onal   Regula4on DNA   Methyla4on 60 Friday, January 13, 2012
  18. 63.

    Phenotype Gene  Copy   Number Sequence   Varia4on Chroma4n  

    Modifica4on Gene   Expression Transcrip4onal   Regula4on DNA   Methyla4on 60 Friday, January 13, 2012