?????:???????????DNA????????,??????????,??????????????????????????????,???????????

1
??????????
2
????

• ???????
• ??????????
• ???????????????
• ?????????????

3
???????

• ????????????????DNA????????,??????????,??????????
  ????????????????????,???????????
• High-throughput Sequencing
• Next Generation Sequencing
• Deep Sequencing

4
???????
??????,???????
????
PCR??
???????
???
A Sanger?? B ?????
5
????

• ???????
• ??????????
• ???????????????
• ?????????????

6
?????????????

• 1992?Lynx Therapeutics MPSS
• 2003?Polony Sequencing(??)
• 2005?454 Pyrosequencing
• 2006?Solexa Sequencing-by-Synthesis
• 2007?ABI SOLiD
• 2008?Helicos tSMS Sequencing
• 2010?Ion torrent Semiconductor Sequensing
• 2011?Pacific Biosciences SMRT Sequensing

7
?????????????
Lynx MPSS
Polony Seq
454
Solexa
Roche 454
ABI SOLiD
Illumina Solexa
Helicos
Ion Torrent
ABI Ion Torrent
SMRT
8
?????????????
????? ???? ???
Roche 454 ????? Roche
Illumina Solexa ?????? Illumina
ABI SOLiD ?????????????? ABI
Helicos ??????? Helicos
Ion Torrent ????? ABI
SMRT ??????? Pacific Bio
9
454 Pyrosequencing

• ??????????

A ??????
C 454????
B 454???
10
454 ????
11
454 ?????Base Calling
12
454 ????????

• ????,400-600bp
• ????,1Run 1M ??,400-600Mb
• ??????
• ????de novo??

13
Illumina Solexa??

• ??PCR
• ??????
• ?????

HiSeq 2000
14
Illumina Solexa ????
15
Illumina Solexa ??PCR
diol
diol
1st cycle denaturation
16
Illumina Solexa Base Calling
17
Solexa ????????

• ????,100-150bp
• ???,25G??,120-150G?Run
• ????RNA??????????

18
ABI SOLiD ??

• SOLiDSequencing by Oligo Ligation/Detection
• Oligo???????????,??oligo?????????

SOLiD 5500xl
19
ABI SOLiD??????
A ????? ????
B ??PCR 3????
C ???? ??????
20
ABI SOLiD????
21
ABI SOLiD?????????
_at_SRR029969.1 VAB_5551_12_381_F3
length35 T11.0203.3.1113211010332111302330201 SR
R029969.1 VAB_5551_12_381_F3 length35 !36!8/8!!
462gt_at_6(lt8gt8.lt29748078 _at_SRR029969.2
VAB_5551_13_468_F3 length35 T202312302.3333130131
131322113203131 SRR029969.2 VAB_5551_13_468_F3
length35 !9),4/3)!((573(96,'791gt)43),(95,
B. SOLiD ??????(Color Space)
A. SOLiD Oligo???????
22
SOLiD ????????

• ????,50-75bp
• ???,??Q40
• ???, 20-30G??,1Run ??120G
• ???????????SNP???

23
?????????
?? 454 Solexa SOLiD
PCR ????PCR ??PCR ????PCR
???? ?? ?? ??
???? ?????? ???????? ??????
???? FastQ FastQ CSFastQ
24
???????????
? ? ?? ?? ?? ??
Solexa HiSeq 2000 Single-end 1 x 35 bp Paired-end 2 x 50 bp Paired-end 2 x 100 bp 25 Gb/d 1.5d 4d 8d 50 bp 85?? Q30 100 bp 80?? Q30
SOLiD 5500xl Single-end 75 bp Paired-end 75 x 35 bp Mate-pair 60 x 60 bp 20 30 Gb/d 1d/ 1lane 7d/ 12 lane 7d/ 12 lane Q40
454 GS FLX 400 - 600 bp 400 600 Mb/Run 10h Q20
25
????

• ???????
• ??????????
• ???????????????
• ?????????????

26
?????????

• DNA??????de novo????????????????????????
• RNA????????RNA?????????
• ???????ChIP-SeqDNA?????

27
?????

• ?????????????DNA??????????,????????????????de
  novo??????????????
• De novo ??????????????????????????,???????????????
  ??????,??????????????
• ???????????????????????????????,??????????????????
  ??

28
???????
Paired-End
Mate-End
???????-??????
29
Paired-end ??
30
Paired-end ???????
31
Paired-end?????????????
Jun Wang, et al. Nature 456, 60-65(6 November
2008)
32
?????????????

• ???????????Base Calling\?????????????????
• ??????????????????????????
• ?????Coding Gene???RNA????????????
• ??????GO?????Interpro?????
• ????????????SNP/InDel/CNV????

33
References

• 1?Erin D. Pleasance, Philip J. Stephens, Sarah O
  Meara, et al.. A small-cell lung cancer genome
  with complex signatures of tobacco exposure.
  Nature, 2010, 463184-190.
• 2?Michael James Clark, Nils Homer, Brain D. O
  Connor, et al.. U87MG Decoded The Genomic
  Sequence of a Cytogenetically Aberrant Human
  Cancer Cell Line. PloS Genetics, 2010,
  6(1)e1000832.
• 3?Wei Chen, Reinhard Ullmann, Claudia Langnick,
  et al.. Breakpoint analysis of balanced
  chromosome rearrangements by next-generation
  paired-end sequencing. European Journal of Human
  Genetics, 2010, 18 539-543.
• 4?Van Tassell CP, Smith TP, Matukumalli LK,
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  sequencing and variant discovery in C. elegans.
  Nat Methods, 2008, 5(2) 183-188.
• 5?Jun Wang, Wei Wang, Ruiqiang Li, et al.. The
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  Nature 456, 60-65(6 November 2008)
• 6?Huang SW, Li RQ, Wang J, et al. The Genome of
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• 7?David Hernandez, et al. De novo bacterial
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  assembled on a desktop computer. Genome
  Res. 2008.18 802-809

34
??????????

• Erin D. Pleasance, et al. The compendium of
  somatic mutations in a small-cell lung cancer
  genome. Nature, 2010, 463184-190.
• ????????????????????NCI-H209????????,?????????????
  ?????????????????????????

35
????????????
36
??????CNV??
37
?????????

• David Hernandez, et al. De novo bacterial genome
  sequencing Millions of very short reads
  assembled on a desktop computer. Genome
  Res. 2008.18 802-809
• ????Staphylococcus aureus strain MW2?Helicobacter
  acinonychis strain Sheeba?????????????,???????????
  ???

38
????????????
39
????????????
?????????????
40
??????

• ??????????????,???????????????????????????????????
  ????????????????,?????????????????????????????????
  ???????? ?????????????????,?????????????????
• ???????????????????????16S/18S rRNA???????

41
???????????

• ?????????,??????? DNA ???????????????,????????????
  ???????????????????????????????,?????????????,????
  ???????????? Sanger???,???,????,???,??????????????
  ?

42
??????????????

• ????
• ????????
• ?????????
• ??Profiling table
• ?????(PCA)
• ??????????????
• ????????

43
16S/18S rRNA??????

• 16S/18S rRNA????????????????????????????,?????????
  ,?16S/18S rDNA??????????,????????,????????????????
  ,?????????????

44
16S/18S rRNA????????

• ???????????
• OTU(Operational Taxonomic Units )??
• ?????
• ??????
• ?????????

45
References

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• George I et al. (2010). "Application of
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• Wong D (2010). "Applications of Metagenomics for
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•  Nelson KE and White BA (2010). "Metagenomics and
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• CharlesT (2010). "The Potential for Investigation
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• Zheng, Hao Wu, Hongwei (2010). "Short
  prokaryotic DNA fragment binning using a
  hierarchical classifier based on linear
  discriminant analysis and principal component
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  9951011.

46
??????????

• ????????????????,??????????????????,??????????????
  ?????? ??????????,????????????????DNA,????????
  ?????,???????????

47
????????????
48
??????????????
49
??????????
???SNP???
??InDel??
50
References

• 1?Wei X, Walia V, et al. Exome sequencing
  identifies GRIN2A as frequently mutated in
  melanoma. Nat Genet. 2011 Apr 15. Epub ahead of
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• 2?Janel O. Johnson, J. Raphael Gibbs,et al. Exome
  Sequencing in Brown-Vialetto-Van Laere Syndrome.
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• 3?Teer JK, Mullikin JC.Exome sequencing the
  sweet spot before whole genomes. Hum Mol
  Genet. 2010 Oct 1519(R2)R145-51. Epub 2010 Aug
  12.
• 4?Ley TJ, Mardis ER, Ding L, et al. DNA
  sequencing of a cytogenetically normal acute
  myeloid leukaemia genome. Nature 2008
  456(7218)66-72
• 5?Gnirke A, Melnikov A, Maguire J, et al.
  Solution hybrid selection with ultra-long
  oligonucleotides for massively parallel targeted
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• 6?Murim Choia, Ute I. Scholla, Weizhen Jia, et
  al. (2010) Genetic diagnosis by whole exome
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• 7?Sarah B Ng, Kati J Buckingham, Choli Lee, et
  al. (2010) Exome sequencing identifies the cause
  of a mendelian disorder. Nature Genetics 42, 30 -
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51
????????????

• Wei X, Walia V, et al. Exome sequencing
  identifies GRIN2A as frequently mutated in
  melanoma. Nat Genet. 2011 Apr 15. Epub ahead of
  print
• ????????????,????????????????????,???????????????

52
???????????????
53
???????????
??GRIN2A???,????????
54
???????

• ?????????????????????????RNA???,??mRNA????RNA(Non-
  coding RNA)?  ????????????????,???????????
  ????,????????????????????????????????mRNA?????,???
  ???UTRs?????????????????????????????cSNP(????????
  ???)????

55
?????????
56
?????????
?????????
?????????
57
?????????
????? ??????? ????? ???????
1 ????????,????????? 2 Contig?Scaffold???? 3 Unigene??????????,GO??,Pathway??,?????? 4 ?????????,??????GO??? Pathway????? 1 ??????,?????? 2 ?????????? 3 ????????????????????? 4 ?????,?????????????????
58
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• Guojie Zhang, Guangwu Guo, Xueda Hu, et al. Deep
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  reveals high complexity of the rice
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• Murchison EP, Tovar C, Hsu A, et al. The
  Tasmanian devil transcriptome reveals Schwann
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  Science. 2010 Jan 1327(5961)84-7.
• Brain B. Tuch, Rebecca R. Laborde, Xing Xu et al.
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• Fuchou Tang, Catalin Barbacioru, Ellen Nordman et
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• Sohrab P. Shah, Ryan D. Morin, Jaswinder Khattra
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  Nature, 2009, 461 809-813
• Zhao et al. Transcriptome-guided characterization
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• Gregory R, Darby AC, Irving H, et al. A de novo
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• Crawford JE, Guelbeogo WM, Sanou A, Traoré A,
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59
????????????

• Maher CA, Kumar-Sinha C,  Cao X, et al.
  Transcriptome sequencing to detect gene fusions
  in cancer. Nature, 2009 Mar 5458(7234)97-101.
• ?????454?Solexa?????????????????VcaP?LNCaP???????,
  ???????????????????????

60
??????
????????
MIPOL1-DGKB ??????
61
????????????

• Crawford JE, Guelbeogo WM, Sanou A, Traoré A,
  Vernick KD, et al. (2010) De NovoTranscriptome
  Sequencing in Anopheles funestus Using Illumina
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  doi10.1371/journal.pone.0014202
• ??????3????????????,???????,????????????????

62
De novo ????????????
??????
????????????
63
???????
??????
??????GO??
??????????????????
64
???????

• ??????????????????????,?????????????????????
• ???????(Digital Gene Expression,
  DGE)???????????(mRNA tag profiling),??????????????
  ???????21nt???????????????????????????,??????????,
  ????????????????????
• ??Tag-SAGE

65
??????????
NlaIII?????
66
?????????

• ??????????????
• ???????,?????????
• ????????,????????????????
• ?????????
• ??????????
• GO??????????
• Pathway????????
• ???????????
• ??????????????

67
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???????????

• Morrissy AS, et al. Next-generation tag
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• ??????????????(Tag-SAGE)???LongSAGE?????????????,?
  ???????,???????????????????,????????,??GC???

69
??????????????
70
GC??????????????
71
?RNA??

• ? RNA?????21-31nt??????????RNA,??????????????,??mR
  NA?????????????????????
• ????RNA???????RNA (miRNA),???RNA(siRNA)??piwi????
  ?RNA(piRNA)?
• miRNA???2124nt,?????????????????(pri-miRNA),?????
  ??mRNA???????????????siRNA,???1925nt,??????RNA,??
  ???????mRNA???????????????piRNA,??2631nt,????????
  Piwi????,????????????????????

72
?RNA?????
73
?RNA??????

• ??????????,?????????,??????,???????
• ????Small RNA?????miRNA / siRNA /
  piRNA????miRNA?? ????miRNA?????

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?RNA??????

• Eugene Berezikov, Nicolas Robine, Anastasia
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• ????????miRNA??????,??????????????,????????miRNA??
  ????????

76
???????MiRNA????
77
MiRNA??????
78
?miRNA??
79
miRNA?????????
80
ChIP-Seq

• ChIP-Chromatin Immunoprecipitation????????,???????
  ?????,???????????????,??????????,????,???????????D
  NA???
• ChIP-Seq??????????ChIP??????????,???????DNA???????
  ??

81
ChIP-Seq????
82
ChIP-Seq????

• ChIP Sequencing??????????????
• ChIP Sequencing reads ????????????reads ?repeats 
  ?????????reads ????????????????reads ?????????
• ????peak ??peak ??peak ??????peak ????????peak
   ?????????????
• Peak?????????GO??????
• ???????????peak ???????????peak ?????

83
ChIP-Seq????
????
????
Unique Mapped ??????
????
Genome Browser???
Peak ??
Peak ??
Peak????
GO????
?????????
84
ChIP-Seq ??????
85
ChIP-Seq??????
86
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87
DNA?????

• DNA??????????????????????,??????????????????,?????
  ?DNA?????????????????
• ??????????DNA????????????,???MeDIP,????DNA????????
  ???,???????,?????DNA???????????Bisulfite
  Sequencing,???Bisulfite??????????????

88
MeDIP ??
89
MeDIP-Seq????

• 1. MeDIP-seq ??????????
• 2. MeDIP-seq ??????????????2.1 MeDIP-seq ??reads 
  ???????????????2.2 MeDIP-seq ??reads ???????????
  2.3 MeDIP-Seq ??reads ?CG?CHG?CHH????????2.4 MeDI
  P-Seq ??reads ?????????????2.5 MeDIP-Seq ??reads 
  ???OE????????
• 3. ??MeDIP-seq ??????(peak)??? 3.1 Peak ??3.2 Pe
  ak ???????????3.3 ????Peak ?OE?????? 3.4 ??Peak 
  ????3.5 ??Peak ?????????????
• 4. ??Peak ?????????4.1 ????????Peak ??????4.2 ??
  ???????????GO???????pathway ????

90
Bisulfite Sequencing??
91
Bisulfite Sequencing????

• 1. Bisulfite-seq??????????
• 2. ????????2.1 C?????????????2.2 ??reads ???????
  ?????
• 3. ??C????????
• 4. ???????????????4.1 ???C???CG,
  CHG ?CHH?????(HA?C or T,???)4.2 CG?CHG?CHH????C?
  ?????4.3 ??????CG?CHG?CHH?C??????(??????????)4
  .4 ?????????CG?CHG?CHH?C??????4.5 ?????????CG?CHG
  ?CHH?C??????4.6 CHG,CHH????C???9bp?????????
• 5. ????DNA ?????5.1 ?????????C???????(??????????
  )5.2 Scaffold????C??????(??????????)5.3 ??????
  ?????????5.4 ???????????DNA????? 
• 6. ???????(DMR)??

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93
????

• ???????
• ??????????
• ???????????????
• ?????????????

94
??????????????

• ????????Perl / BioPerlPython / BioPythonR /
  BioconductorJAVA / BioJava
• ??????NCBI SRA Sequence Read ArchiveUCSC
  Genome BrowserSEQanswers WiKi Forum for NGS

95
?????????

• Velvet
• Ray
• ABySS
• SOAPdenovo
• SSAKE
• SHARCGS
• MIRA
• Edena

96
???????

• BLAST
• BLAT
• MAQ
• SOAP
• Bowtie
• BWA
• SSAHA
• ELAND

97
SNP ????

• SAMTools
• SOAPsnp
• NGS-Backbone
• MAQ
• SeqMan NGen
• CLCBio Genomics

98
????!