Application of Bioinformatics on Cancer Research

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4. Internet?????????

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Blast
Blast
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How is tumor generated ?
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1996, 97????????
??????????????,????? VS IBM??
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What Can Bioinformatics Do in Cancer
Research?How to Do?
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?????(??Linux???MySQL?????)

• 1) Reference, Locus Link, Unigene,
  Mapview???GenBank????
• 2) Gene Ontology ???, ?????,????,?????????????????
  ????????????

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?????(?)

• 3) UCSC Human Genome ??? (Golden Path)?
• 4) ????Blast??? (nt, nr, human_est, htg,
  swissprot, yeast, mouse_est )?
• 5) ????????????????(????)

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Computational Velocity Doubled after 18
monthsDNA Data Quantity Doubled after
14 months
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???????????

• cDNA, mRNA?EST????????????
• ??????????
• ???? (Gene-gene interaction)????

????????????,?????????????????
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cDNA, mRNA?EST????????????

• a) ?????,??????,????????,?????????,?Fasta?????
• b) ??Reference mRNA???Unigene??,??????,????????
• c) ?????????????????,???????????,???????

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cDNA, mRNA?EST???????????? (?)

• d) ?EST????,??cDNA???????????????
• e) ?????SNP??
• f) ???????? (PDF??)

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Primary Analysis of Lung Cancer SSH cDNA Library
????
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Definition of EST
????
EST (Expressed Sequence Tag) ?cDNA
???????????????,?????500bp?

• ESTs offer a rapid and inexpensive route to gene
  discovery, reveal expression and regulation data
  (Vasmatis, et al,1998), highlight gene sequence
  diversity and splicing (Wolfberg and Landsman,
  1997), and may identify more than half of known
  human genes (Hillier, et al, 1996).

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Procedures
Sequencing Result
Mask Vector and Format
Blast to Reference mRNA DB
Reference DB None-hit EST
Blast to Human EST DB
Human EST DB None-hit EST
Screened Known Genes
Cluster ESTs by Gene
Blast to Human Genome
Map to Human Genome
New Genes
Garbage ESTs
Gene Expression Map
Point Mutation/SNP Analysis
In silico EST Elongation
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Original sequence Database
??????
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Cluster Result Database
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???EST
New Gene (EST) Database
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Elongated EST
???EST??????

• gtIDNo2_rlcrt0-000159.fasLength2540
• ......AGCGGGTCCCGCCTCCCAGCGACTCTCGGCAGTGCCGGAGTCGG
  GTGGGTTGGCGGCTATAAAGCTGGTAGCGAAGGGGAGGCGCCGCGGACTG
  TCCTAGGTACACTTTTCTCATAAAGTTTAGCCTACAGAAACTATCGCCAC
  CCAAATTAAACATCACCCAAGCTAATATTCTTTCCTCCTTCTAAAGATGA
  GCTAGCGAAACTTTTTATAGGTTGTCCCTTTAATGCAGCTTTTTAGAATA
  AACATTTTTACATTTTTTCTTAAAAGAATTATTTTTTGAAGTCTGAGGAA
  AAATCCGCTTGCCTAGTGAATTTGGCACACACAGAGTAACAACAAATCAA
  ACTTTAAGCTAGCAACCAACACACAAAATAAGCATGCAAGGAATAGAATA
  AGTTTTATATGGATAAGGTATTTTAGCCAACTCCACTTATAAGGTATTAC
  AAAATCTCTATATNGTTTTGAAGCTATGTGTCGCAGTTTAAAGTTACTTT
  TAACAATAATACGTATATTTACAATTGACTTAAAAAACTATTTTCAAGGA
  AGTTAGAAACCTATGGCACACCAACGCATCTTCTGGAAAATGAAGACGAT
  ACAATGTCATGTGGCAAGTTTCAATATATGAAGGACTAGACCAGTG....
  ..........

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Using Reference mRNA Database Blast Output to
Search Mutation/SNP
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Mutations Found from Blast Output Analysis
???/SNP????
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Deletion (ClustalX 1.82)
???/SNP???? (?)
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???/SNP????
Insertion (ClustalW 1.82)
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???/SNP???? (?)
Here - means insertion
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(No Transcript)
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Further Analysis

• From SNP to Haplotype

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6 SSH Libraries Gene Expresstion Map to Human
Genomes(122X)
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Expression level of genes in SSH libraries
Different Colors correspond to different libraries
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Is there a LOH?
Expressed in two down-regulated libraries
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LOH map vs. SSH map
Lung Cancer Related LOH
Lung Cancer Related SSH
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??????????

• a) ?raw data???????,???thredshold?(???????????,???
  ???)
• b) ??R/S,SAS???????????????????????
• c) ??????????????,???????????????????????????arra
  y?????????,??????????(hierarchical, SOM and
  K-means clustering) ??Gene Ontology, Biocarta,
  KEGG???????pathway???

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Normalization
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PathWay Analysis
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Genome-wide Gene Expression Map and Analysis of
Non-Small Cell Lung Cancer Based on Microarray
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PNAS November 20, 2001 vol. 98 no. 24
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Original Array Data

• Chip Human U95A oligonucleotide probe arrays
  (Affymetrix, SantaClara, CA) 12,600 cDNA clone
• Sample
• Squamous cell lung carcinomas 21
• Adenocarcinomas 127
• Normal Lung 17
• Array data (normalized)

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Analysis Procedures

• Finding genes from 12,600 cDNA clone
• Get 7932 genes
• Flagging the values lower than threshold value
• About half values keeped
• Doing T-test with SAS/R for each gene
• Hierarchical Clustering
• Divide into two parts up-regulated and
  down-regulated
• Construction of Gene Expression Map and
  Transcriptome Map

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Clustering Result
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?????????(3????)
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Analysis Procedures (Cont.)

• High-resolution detection of differentially
  expressed chromosomal regions in NSCLC was
  obtained by using moving-median method
• Screening of important NSCLC-related gene

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Results
???????????????

• 75 (24 of 32) of our results were consistent
  with the previous studies. And the counterparts
  in other reports, normally with larger sizes,
  were narrowed down and many specific genes
  involved in these regions were identified.
• 4 new aberrant regions in squamous carcinoma,
  2q31-32, 12q23-24, 14q22-q24 and Xp11.4-p11.23,
  were discovered.

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???? (Gene-gene Interaction) ????

• a) ?????,? GO??????????,?? extracellular???????
• b) ??GO, BioCarta?Kegg???????????????????,?????
• c) ?????????,????????????,?????????????
• d) ????????(????

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Gene Ontology Pathway Network
??DAG (???), ???????
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BioCarta Cell Cycle Pathway
?????????
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KEGG ???? Pathway
?????????
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????

• GO0003673-gtbiological_process-gtphysiological
  processes-gtcell growth and/or maintenance(D10S170)
  -gtcell proliferation(FTH1,AKR1C3,C20orf1)-gtcell
  cycle(AHR,BUB1,STAG2)-gtDNA replication and
  chromosome cycle-gtchromosome segregation(STAG2)-gtm
  itotic chromosome segregation

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???????????????????,?????????????
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• 26 N 15 219 T 78 429 nucleotide binding
• 32 N 28 396 T 120 728 nucleic acid binding
• 2 N 1 31 T 21 91 structural constituent of
  ribosome
• 47 N 0 0 T 1 7 apoptosis inhibitor activity
• 38 N 0 0 T 10 50 transcription factor activity
• 37 N 1 13 T 1 13 enzyme inhibitor activity
• 46 N 6 46 T 6 46 metal ion binding

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?????????????

• 1) ??mRNA/cDNA???siRNA??????siRNA???????,?????
  Human Genome???????,????????????????
• 2) ??????????,?????? cDNA?????cDNA??(image
  clone)???
• 3) DNA???????cDNA????????????????????????

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?????????????(?)

• 4) ????????????????? (????)???,?????????,?Gene
  Ontology??????????????domain??????????
• 5) ????accession number,??????????????????????,???
  ????????

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?????????????(?)

• 6) ???SAGEmap??,??????????NCBI SAGEmap????????????
  
• 7) DNA/RNA ??ORF?????,???????,cDNA?EST???ORF??????
  ???
• 8) ??EST???????cDNA???

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?????????????(?)

• 9) ???????????,????????????,????????????????????ov
  erlap??????,?????????????
• 10) ??Blast??????
• 11) ???????contig????????????????,????????

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????
??Gene Ontology????EMBOSS?????SSH?????????(??2???
?)
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Screened siRNA target sites for X1blue
????

• gtIDX1_blueNonsense0Length21GC38A8G5C3
  T5
• AAAGATGTGGAAAGTTACCTC
• siRNA
• Sense AGAUGUGGAAAGUUACCUC
  UU
• Antisense GAGGUAACUUUCCACAUCU
  UU
• Negative Control Sense GGAUGUACGGCAAAUUCUAUU
  
• Negative Control Antisense UAGAAUUUGCCGUACAUCCUU

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???SAGEmap??
????
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?accession number??????????????????????
????
gtNM_005400 Homo sapiens protein kinase C, epsilon
(PRKCE), mRNA. CTCCCCGCCCCGACCATGGTAGTGTTCAATGGCCT
TCTTAAGATCAAAATCTGCGAGGCC GTGAGCTTGAAGCCCACAGCCTGG
TCGCTGCGCCATGCGGTGGGACCCCGGCCGCAGACT TTCCTTCTCGACC
CCTACATTGCCCTCAATGTGGACGACTCGCGCATCGGCCAAACGGCC.
gtNM_005813 Homo sapiens protein kinase C, nu
(PRKCN), mRNA. AAAGTTCATCCCCCCAGAATGAAAATGAGGACATT
TGAGAAGGTGATTTAAGGTGTGGAC ATTTGAGAAGGTGTCCTATCAAAT
TAGTAAACCAAAGGAAAAGTACTGAATAGATTAATC gtHSPKCB2A
Human mRNA for protein kinase C (PKC) type beta
II. CAGAGCCGGCGCAGGGGAAGCGCCCGGGGCCCCGGGTGCAGCAGCG
CCCGCCGCCTCCCG

• NM_002737
• NM_002738
• X07109
• NM_002739
• NM_002740
• NM_006255
• NM_005400
• NM_002742
• NM_005813
• L07032
• NM_002744
• NM_006254

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??????????????

1. EMBOSS (????????????,?????Linux?????GCG???,???????
   ? )
2. JaMBW (Java based Molecular Biologists
   Workbench)???????????,?????? European Molecular
   Biology Laboratory of Heidelberg?Java????????????,
   ???????????????,????,???? ?

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??????????????(?)

• SMS (The Sequence Manipulation
  Suite)???DNA???????????????,????????
  ???????????HTML??,??????????,????HTML?????????
• PredictProteinProtein sequence analysis, and
  structure prediction.
• GeneScan ?????genomic DNA??????????
• RepeatMasker ??????

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??????????????(?)

• Cross_match/Consed/phred/Phrap/phd2fasta
  ???????,?????????????,??Contig ???????
• Primer 3 ??web?PCR???????
• sim4 EST?????????
• HMMER ???????????(HMM)?????????,?????????????????
  ?
• ClusterW/X ??????????
• Artemis DNA????????????
• BioPerl ?????????Perl???

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• 2000?????????
• 2001?4?????? HP-LH3000
• 2001?9???????????????????(????????????
  24??),??????
• 2002????????????????

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????????? http//bioinfor.cicams.ac.cn
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• ???
• ????? ?????
• ???? ???520??
• ???? 67781331-8665
• Email xyzh_at_263.net.cn
• ??http//bioinfor.cicams.ac.cn

?????????????????.doc??, ????8665???email?
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Thanks !