Outline

1
Outline

• Biology 101-Cells, DNA, DNA replication
• Genes, EST
• Genetic markers, SSR
• Bioinformatics
• Database, Blast
• Predict SSR from public EST sequences
• Goal, Method and Results

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Cell-Smallest unit of Life

• Basic unit of life
• Organisms
• Single Cell
• Colonies/clusters
• Multicellular (cell differentiation)
• Multicellular organisms
• cell ? tissue ? organ ? system ? organism

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Chemical Foundation for Cells

• Organic compound molecules of life
• consist of atoms of C, H, O and sometimes other
  elements
• Carbohydrates starches, sugars, cellulose -
  used for energy and building material
• Lipids store fat for energy
• Protein building material, structural, enzyme
• Nucleic Acids DNA, RNA, basis of inheritance and
  cell reproduction

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Cell Structure and Function

• Nucleus DNA, RNA, replication
• Ribosomes protein synthesis
• Endoplasmic reticulumprotein, lipid, others
  synthesis
• Golgi apparatusprocessing, packaging, secretion
  of proteins
• Mitochondrion cellular respiration
• Chloroplasts (plant only) Photosynthesis
• Vacuoles Vesicles storage of substances
• Lysosomes intracellular digestion

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DNA, Chromosome and Gene

• DNA An enormous double-stranded, twisted
  molecule densely coiled around molecular beads of
  histone protein to form a chromosome.
• Genes A strand of DNA from a chromosome with
  very long series of coded messages.
• Each gene is composed of many thousands of
  letters called basesA, T, G, C, (A-T and G-C are
  pairs).
• In order for the cell to decipher the DNA strand,
  the bases must be read in groups of 3 letters
  called codons.
• It is also important to start reading on the
  correct letter of the base sequence where the
  first codon of the gene begins.
• Since there are four different possibilities for
  each base of a codon, the total number of DNA
  codons is 43 or 64.
• Some codons represent "punctuation marks" marking
  the beginning or the end of a gene message.

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• LSHTHEMANANDBOYARESAD THE MAN AND BOY ARE SAD
• AUGAATCGCATACTAAGACAG AAT CGC ATA CTA AGA CAG
• Point mutation
• AATCGCATACTAAGATAG
• THEMANANDBOYAREBAD
• DNA

Start codon
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DNA-gtRNA-gtProtein
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DNA Replication

• DNA replicates before cell division
• 2 complementary strands serve as template for the
  new construction
• Unwind DNA, separate 2 strands by DNA Helicase
• replication origins (characterized by a weak bond
  between the two DNA strands) formed
• A small RNA attached to the single stranded DNA
  by DNA primase
• DNA polymerase copy the DNA template with high
  fidelity
• Only work 5-3 direction, different approach for
  two strands

Replication, leading strand Replication,
lagging strand
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EST-Expressed Sequence Tags

• Genome is big, 600,000-3 billion base pair
• Small fraction of genome, usually expressed gene

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Genetic Markers

• Phenotype (characteristics of individuals or
  different species)
• Genotype (genetic information, DNA sequence)
• Genetic diversity is characterized by genetic
  markers
• DNA sequence difference
• Phenotype difference
• No phenotype difference
• Different type of genetic markers

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Type of genetic markers

• AFLP Amplified Fragment Length Polymorphism
• RFLP Restriction Fragment Length Polymorphism
• Allozymes proteins variants
• cDNA markers coding region of genes
• Microsatellites (SSR-simple repeat sequence)

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SSR

• Molecular marker loci consisting of tandem repeat
  units of very short (1-5 basepairs) nucleotide
  motif
• TTTTTTTTTT, ATATATATATAT
• The number of repeats are different in different
  species and individuals
• In case the nucleotide sequences in the flanking
  regions of the microsatellite are known, specific
  primers (generally 20-25 bp) can be designed to
  amplify the microsatellite by the Polymerase
  Chain Reaction (PCR)
• The size of the amplified fragment is different
  and can be detected by gel-electrophoresis.
• How to identify the flanking region of the SSR?

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• Constructing a small-insert genomic library,
  screening of the library with a synthetic labeled
  oligonucleotide repeat and sequencing of the
  positive clones
• Primers designed for closely related species may
  be used
• Bioinformatics approach-searching the public
  available EST sequence database
• Large amount of EST available
• Fast, large scale analysis
• Lower cost

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What is Bioinformatics

• Study of biological information
• Interface of biology and computers
• Computational molecular biology
• Includes genomics
• Subfields DNA informatics, protein informatics,
  proteomics
• Person is bioinformaticist
• Sometimes bioinformatician, bioinformatist

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What is Bioinformatics

• A narrower definition
• The use of computer tools to store, access,and
  analyze nucleic acid and amino acid sequence data
  and protein structural data.
• A broader definition
• The use of computer tools to store, access, and
  analyze all types of biological data, including
  text data, phylogenetic trees, metabolic maps.
• Many applications in biological research, drug
  design, medicine.

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Bioinformatics Related Fields

• Biotechnology
• Computer simulations in biology
• Genetics, biochemistry, cell biology
• Structural biology
• Phylogenetics and taxonomy
• Quantitative biology, mathematical biology,
  theoretical biology
• Epidemiology
• Medical informatics
• Drug design

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Early History

• First protein amino acid sequence database in mid
  1960s.
• Researchers developing algorithms to analyze
  these data for individual research projects in
  1960s and 1970s.
• GenBank and other public databases with freely
  available analysis tools in the 1980s
• Enormous growth of GenBank and PDB in 1990s

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Recent Trend

• A great surge in genomics
• The Human Genome Project
• Genome projects for 400 organisms
• gt100 completed published genomes
• Recent advances in molecular genetics
  technologies, especially microarrays
• Push to analyze genes and gene products, and to
  determine protein structure/function relationship
• High through-put biology, large scale data
  analysis

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Data Explosion

• These research trends, especially the sequencing
  projects, have resulted in huge amounts of raw
  data that must be stored, manipulated, and
  analyzed.
• Computer information technology has also advanced
  very rapidly and has provided the means for
  handling this data explosion.

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GenBank increase in basepairs and DVA sequence
files
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A very Broad field

• mapping
• evolutionary studies
• sequencing
• statistical analysis of biopolymers
• sequence comparisons
• gene modeling
• DNA sequence -gt DNA structure
• molecular modeling

• structure comparisons
• sequences/structures -gt function
• gene expression and genetic/metabolic networks
• database
• visualization and interaction
• comparing tools
• new technologies
• ethics

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Problems in Bioinformatcs

• Genomics
• Gene finding
• Annotation
• Sequence alignment and database search
• Functional genomics
• Microarray expression, gene chips
• Proteomics
• Structure prediction
• Comparative modeling
• Function prediction
• Structural bioinformatics
• Molecular docking, screening, etc.

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Bioinformatcs Tools and Services

• Databases text, sequence, structure
• Database annotation text searches
• Sequence similarity search tools
• Sequence and structure analysis tools
• 3D structure visualization tools
• Structure prediction tools
• Phylogenetic analysis tools
• Metabolic analysis tools

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Major Bioinformatics Site

• NCBI, The National Center for Biotechnology
  Information http//www.ncbi.nlm.nih.gov
• EMBL, The European Molecular Biology Laboratory
  http//www.ebi.ac.uk
• PDB, Protein Data Bank www.rcsb.org/pdb
• This is just a sample list of the most used sites

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NCBI

• Entrez DNA, protein, genome database
• BLAST Offer blast search against many ncbi
  databases
• PubMed Literature search service that provides
  access to gt10 million citations.
• COGs (Clusters of Orthologous Groups) ancient
  conserved protein domains from the complete
  genomes
• ORF Finder Finds all open reading frames in a
  user or database sequence.
• FTP server for databases and software
• Many other features, including examples and
  tutorials

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What is BLAST

• BLASTBasic Local Alignment Search Tool
• Most widely used search program
• Finds and aligns matching sequences.
• Fast
• Several versions, eg. DNA or protein query
• Current since 1997 BLAST 2.0 gapped BLAST
• Does alignments of local regions, not entire
  sequences.

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Sequence Questions

• Does the DNA sequence contain a gene?
• Is the gene a member of a known gene family? What
  is the encoded protein
• What is the function of the protein?
• Do other organisms have the protein or the gene?

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Sequence Implications

• DNA sequence of gene determine amino acid
  sequence of a protein. Primary sequence
  determines structure and function of a gene.
• Proteins with similar sequences and structures
  have similar functions
• Similar sequences should have long regions of
  identical or similar residues. Why?
• Evolution decent from a common ancestral
  sequence. Homology is implied for each similar
  region
• Very rarely have functional convergence without
  sequence or structure similarity.

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Sequence Similarity Search

• Look for DNAs or proteins with similar sequence
  to query, by searching a sequence database.
• Search requires
• Search software
• Databases of annotated sequences
• Useful output
• Results must be interpreted and evaluated.

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Similarity Search Terms

• A similarity search of a database is performed by
  aligning a single query sequence to search
  sequence in the database in turn. Good matches
  may be found to subject sequences
• Local alignment align local regions of
  sequences. Local alignment returns a list of
  HSPs High-scoring Segment Pairs.

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Alignment Quality

• Features of good alignments
• Many exact matches of residues between sequences.
• Some mismatches which nevertheless are AAs with
  similar physicochemical properties.
• Few gaps.
• Long stretches of very good match.
• Query 162 ---LKFGNMKVETFYPGKGH 181
• FG VE FYPG H
• Sbjct 162 GDAVRFG--PVELFYPGAAH 182

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Different BLASTS

• BLASTN
• Compares a DNA query to DNA database
• Searches both strands automatically
• Is optimised for speed, not sensitivity
• BLASTP
• Compares a protein query to protein database
• BLASTX
• Compares a DNA query to protein database
• Does 6-frame translations of the query
• TBLASTN
• Compares a protein query to a DNA database
• Does 6-frame translations of the database

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Different BLASTS

• TBLASTX
• Compares a DNA query to a DNA database
• Does 6-frame translations of the query and of the
  database
• Each search is comparable to 36 BLASTP searches!
• BLAST2
• Also called 'advanced' BLAST
• Can perform gapped alignments

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The pipeline to generate potential SSR markers
from the EST sequence
EST sequences
Output of results 1. Position, type, of repeats
of identified SSR 2. Flanking primers
sequences, melting T, product size, etc 3.
Annotation by searching TGI
Pre-processing (remove polyA/T, etc)
SSR search Identification of SSR, Definition of
minimum number of repeats N(10), NN(6), NNN(5),
NNNN(5), NNNNN(5), NNNNNN(5)
Primer3-Primer prediction
CAP3-assemble the ESTs Remove redundancy
Lab work to confirm the predicted SSR markers
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Download EST from genbank
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Clean Up the EST sequences

• Trim the poly A, or poly T sequences
• Remove the low quality sequences (if there is a
  lot of Ns)
• Remove sequence gt 700 bp in a EST
• Remove the very short sequences lt100 bp
• EST 94,423 to 94,340 sequences

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Perl scripts to find the SSR sequence

• Criteria for SSR
• Definement of SSR (unit size / minimum number of
  repeats) (1/10) (2/6) (3/5) (4/5) (5/5) (6/5)
• Maximal number of bases interrupting 2 SSRs in a
  compound microsatellite 100
• For each individual sequences, read base one by
  one, count the number of repeats, if meet
  criteria, output the sequence and the position,
  type of the SSR to one file.
• Results
• Total number of identified SSRs
  6725
• Number of SSR containing sequences
  6051
• Number of sequences containing more than 1 SSR
  600
• Number of SSRs present in compound formation
  39

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Primer3 to design the primer

• Primer3, a free available software to design the
  PCR primer based on DNA sequence
• Can define the primer pickup criteria
• Results
• 5045 SSR sequences were successfully predicted
  with primer pairs
• 1282 SSR sequences failed

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Use CAP3 to assemble the SSR containing EST
sequence to avoid redundancy

• CAP3, a free available software to assemble DNA
  sequences based on sequence similarity
• CAP3 assemble the EST sequences using BLAST
• BLAST most heavily used searching tool
• Blastn, blastp, blast2
• Use public available database nr, nraa, est,
  human, mouse, rat
• Use customized database

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All the ESTs represent same part of DNA sequence
in this figure
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Summary of Statistics

• Total number of sequences examined
  94340
• Total size of examined sequences (bp)
  50197414
• Total number of identified SSRs
  6725
• Number of SSR containing sequences
  6051
• Number of sequences containing gt1 SSR 600
• Number of SSRs present in compound formation
  398
• Unit size Number of SSRs
• 1 3141
• 2 805
• 3 2652
• 4 53
• 5 10
• 6 64