Data Acquisition Tools

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• Data Acquisition Tools Techniques

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In this presentation

• Part 1 Sequencing Technology
• Part 2 Genomic Databases

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Part1

• Sequencing Technology

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Principles of DNA sequencing

• DNA sequencing is performed using an automated
  version of the chain termination reaction, in
  which limiting amounts of dideoxyribonucleotides
  generate nested sets of DNA fragments with
  specific terminal bases
• Four reactions are set up, one for each of the
  four bases in DNA, each incorporating a different
  fluorescent label
• The DNA fragments are separated by PAGE and the
  sequence is read by a scanner as each fragment
  moves to the bottom of the gel

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Types of DNA sequencing

• DNA sequences come in three major forms
• Genomic DNA comes directly from the genome and
  includes extragenic material as well as genes.
  In eukaryotes, genomic DNA contains introns
• cDNA is reverse-transcribed from mRNA and
  corresponds only to the expressed parts of the
  genome. It does not contain introns
• Recombinant DNA comes from the laboratory and
  comprises artificial DNA molecules such as
  cloning vectors

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Genome sequencing strategies

• Only short DNA molecules (800 bp) can be
  sequenced in one read, so large DNA molecules,
  such as genomes, must first be broken into
  fragments. Genome sequencing can be approached in
  two ways
• Shotgun sequencing involves the generation of
  random DNA fragments, which are sequenced in
  large numbers to provide genome-wide coverage
• Clone contig sequencing involves the systematic
  production and sequencing of subclones

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Sequence quality control

• High quality sequence data is generated by
  performing multiple reads on both DNA strands
• Preliminary trace data is then base called and
  assessed for quality using a program such as
  Phred
• Vector sequences and repeated DNA elements are
  masked off and then the sequence is assembled
  into contigs using a program such as Phrap
• Remaining inconsistencies must be addressed by
  human curators

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Single-pass sequencing

• Sequence data of lower quality can be generated
  by single reads (single-pass sequencing)
• Although somewhat inaccurate, single-pass
  sequences such as ESTs and GSSs can be generated
  in large amounts very quickly and inexpensively

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RNA sequencing

• Most RNA sequencing are deduced from the
  corresponding DNA sequences but special methods
  are required for the identification of modified
  nucleotides. These include biochemical assays,
  NMR spectroscopy and MS

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Protein sequencing

• Most protein sequencing is now-a-days carried out
  by MS, a technique in which accurate molecular
  masses are calculated from the mass/charge ration
  of ions in a vacuum
• Soft ionization methods allow MS analysis of
  large macromolecules such as proteins
• Sequences can be deduced by comparing the masses
  of tryptic peptide fragments to those predicted
  from virtual digests of proteins in databases
• Also, de novo sequencing can be carried out by
  generating nested sets of peptide fragments in a
  collision cell and calculating difference in mass
  between fragments differing in length by a single
  amino acid residue

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Importance of protein interactions

• They underlie most cellular functions.
  Protein-protein interactions result in formation
  of transient or stable multi-subunit complexes
• Understanding of these complexes is required for
  functional annotation of proteins and is a step
  towards the elucidation of molecular pathways
  such as signaling cascades and regulatory
  networks
• Protein interactions with nucleic acids form an
  important area of study, since such interactions
  are required for replication, transcription,
  recombination, DNA repair and many other
  processes. Proteins also interact with small
  molecules, which act as ligands, substrates,
  cofactors and allosteric regulators

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Methods for protein interactions

• Genetic methods
• Suppressor mutant
• Synthetic lethal effect
• Dominant negative mutations
• Affinity methods
• Affinity chromatography
• Co-immunoprecipitation

• Molecular and atomic methods
• X-ray crystallography
• NMR spectroscopy
• Other methods
• FRET
• SPR spectroscopy
• SELDI
• Library-based methods
• Y2H system

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Other methods

• For larger proteins that do not readily form
  crystals, alternative analytical methods are
  required to deduce structures
• These include X-ray fiber diffraction, electron
  microscopy and circular dichroism (CD)
  spectroscopy

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Protein structure determination

• X-ray crystallography
• NMR spectroscopy
• Other methods
• X-ray fiber diffraction
• Electron microscopy
• CD spectroscopy

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X-ray crystallography

• Involves determination of protein structure by
  studying diffraction pattern of X-rays through a
  precisely orientated protein crystal
• They way in which X-rays are scattered depends on
  the electron density and spatial orientation of
  the atoms in the crystal
• A mathematical method called the Fourier
  transform is used to reconstruct electron density
  maps from the diffraction data allowing
  structural models to be built

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NMR spectroscopy

• NMR is a property of certain atoms that can
  switch between magnetic states in an applied
  magnetic field by absorbing electromagnetic
  radiation
• The nature of absorbance spectrum is influenced
  by the type of atom and its chemical context, so
  that NMR spectroscopy can discriminate between
  different chemical groups
• NMR spectra are also modified by the proximity of
  atoms in space
• Analysis of NMR spectra allows 3D configuration
  of atoms to be reconstructed, resulting in a
  series of structural models
• The technique is suitable only for the analysis
  of small, soluble proteins

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2-D gel electrophoresis

• The current method for studying proteins consists
  in part of a technique called two dimensional gel
  electrophoresis, which separates proteins by
  charge and size
• In the technique, researchers squirt a solution
  of cell contents onto a narrow polymer strip that
  has a gradient of acidity. When the strip is
  exposed to an electric current, each protein in
  the mixture settles into a layer according to its
  charge. Next, the strip is placed along the edge
  of a flat gel and exposed to electricity again.
  As the proteins migrate through the gel, they
  separate according to their molecular weight.
  What results is a smudgy patterns of dots, each
  of which contains a different protein
• In academic laboratories, scientists generally
  use a tool similar to a hole puncher to cut the
  protein spots from 2-D gels for individual
  identification by another method, mass
  spectroscopy
• Now-a-days, companies have started using robots
  to do it

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Part2

• Genomic Databases

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Types of databases

• There are many types of databases available for
  researchers in the field of biology
• Primary sequence databases - for storage of raw
  experimental data
• Secondary databases - contain information on
  sequence patterns and motifs
• Organism specific databases
• Other databases

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Primary sequence databases

• Three primary sequence databases are GenBank
  (NCBI), the Nucleotide Sequence Database (EMBL)
  and the DNA Databank of Japan (DDBJ)
• These are repositories for raw sequence data, but
  each entry is extensively annotated and has
  features table to highlight the important
  properties of each sequence
• The three databases exchange data on a daily basis

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Subsidiary sequence databases

• Particular types of sequence data are stored in
  subsidiaries of the main sequence databases. For
  instance, ESTs are stored in dbEST, a division of
  GenBank
• There are also subsidiary databases for GSSs and
  unfinished genomic sequence data

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Organism specific resource

• As well as general databases that serve the
  entire biology community, there are many organism
  specific databases that provide information and
  resources for those researches working on
  particular species
• The number of such databases is growing as more
  genome projects are initiated, and many can be
  accessed from general genomics gateway sites such
  as GOLD

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Organism-specific genomic databases
Organism Database/resource URL
Escherichia coli EcoGene EcoCyc (Encyclopedia of E. coli genes and metabolism Colibri http//bmb.med.miami.edu/EcoGene/EcoWeb http//ecocyc.pangeasystems.com/ecocyc/ecocyc.html http//genolist.pasteur.fr/Colibri
Bacillus subtilis SubtiList http//genolist.pasteur.fr/SubtiList
Saccharomyces cerevisiae Saccharomyces Genome Database (SGD) http//genome-www.stanford.edu/Saccharmyces
Plasmodium falciparum PlasmoDB http//PlasmoDB.org
Arabidopsis thaliana MIPS Arabidopsis thaliana Database (MAtDB) The Arabidopsis information resource (TAIR) http//mips.gsf.de/proj/thal/db http//www.arabidopsis.org
Drosophila melanogaster FlyBase http//flybase.bio.indiana.edu
Caenorhabditis elegans A C. elegans DataBase (ACeDB) http//www.acedb.org
Mouse Mouse Genome Database (MGD) http//www.informatics.jax.org
Human OnLine Mendelian Inheritance in Man (OMIM) http//www.ncbi.nlm.nih.gov/omim
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Finding organism-specific databases

• Organism specific databases are widely
  distributed on the Internet
• In order to find and interrogate databases on
  specific organisms, it is necessary to use a
  gateway site to access relevant databases and
  information resources
• Worked examples are provided, using GOLD as the
  gateway and illustrated with Ebola virus, the
  bacterium E. coli, the fruit fly Drosophila
  melanogaster and the human genome

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Useful gateway sites providing information on
multiple, organism and genomic resources
Gateway site URL
NCBI Genomic Biology www.ncbi.nlm.nih.gov/Genomes/
GOLD (Genomes OnLine Database) wit.integratedgenomics.com/GOLD
Organism specific genomic databases www.unl.edu/stc-95/ResTools/biotools/biotools10.html
TIGR Microbial Database www.tigr.org/tdb/mdb/mdbcomplete.html
Bacterial genomes genolist.pasteur.fr
Yeast database genome-www.stanford.edu/Saccharomyces/yeast_info.html
EnsEMBL genome database project www.ensembl.org
MIPS (Munich Information Centre for Protein Sequences) mips.gsf.de
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Nematode
Bakers Yeast Cells
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Other databases

• Specialized sequence databases for storage and
  analysis of particular types of sequences e.g.,
  rRNA and tRNA, introns, promoters and other
  regulatory elements
• OMIM for study of human genetics and molecular
  biology
• Incyte and UniGene for providing gene sequences
  and transcripts with expert annotation for use in
  drug design and research
• Structural databases for protein structural
  data (e.g. PDB, MMDB) containing X-ray Crys.
  and NMR studies
• Proteins and higher order functions to store
  information on particular types of proteins such
  as receptors, signal transduction components,
  regulatory hierarchies and enzymes
• Literature databases to store scientific
  articles with text search facility (e.g. Medline
  and PubMED)

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Database tools for displaying and annotating
genomic sequence data
Viewer format URL
Artemis www.sanger.ac.uk/Software/Artemis
ACeDB www.acedb.org/Tutorial/brief-tutorial/shtml
Apollo www.ensembl.org/apollo
EnsEMBL www.ensembl.org
NCBI map viewer www.ncbi.nlm.nih.gov
GoldenPath genome.ucsc.edu
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Database formats

• There is no universally agreed format for genome
  databases and several viewers and browsers have
  been developed with graphical displays for
  genomic sequence analysis and annotation
• One of the most versatile formats is ACeDN
  (originally designed for the nematode C.
  elegans), which has an object-oriented database
  architecture and is now used in many applications
  outside the field of genomic bioinformatics

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Common formats

• There are several conventions for representing
  nucleic acid and protein sequences, of which the
  following are widely used
• NBRF/PIR
• FASTA
• GDE
• These formats have limited facilities for
  comments, which must include a unique identifier
  code and sequence accession number

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Formats for multiple sequence alignment

• There are separate formats for multiple sequence
  alignment representation, of which the following
  are popular
• MSF
• PHYLIP
• ALN

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Files of structural data

• Structural data are maintained as flat files
  using the PDB format
• Such files contain orthogonal atomic co-ordinates
  together with annotations, comments and
  experimental details

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Submission of sequences

• Sequences may be submitted to any of the three
  primary databases using the tools provided by the
  database curators
• Such tools include WebIn and BankIt, which can be
  used over the Internet, and Sequin, a stand-alone
  application

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Database interrogation

• All the databases discussed above can be searched
  by sequence similarity
• However, detailed text-based searches of the
  annotations are also possible using tools such as
  Entrez
• The simplest way to cross-reference between the
  primary nucleotide sequence databases and
  SWISS-PROT is to search by accession number, as
  this provides an unambiguous identifier of genes
  and their products

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Databases covered by Entrez
Category Database
Nucleic acid sequences Entrez nucleotides sequences obtained from GenBank, RefSeq and PDB
Protein sequences Entrez protein sequences obtained from SWISS-PROT, PIR, PRF, PDB and translations from annotated coding regions in GenBank and RefSeq
3D structures Entrez Molecular Modeling Database (MMDB)
Genomes Complete genome assemblies from many sources
PopSet From GenBank, set of DNA sequences that have been collected to analyze the evolutionary relatedness of a population
OMIM OnLine Mendelian Inheritance in Man
Taxonomy NCBI Taxonomy Database
Books Bookshelf
ProbeSet Gene Expression Omnibus (GEO)
3D domains Domains from the Entrez MMDB
Literature PubMED
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Databases covered by DBGET/LinkDB
Category Database
Nucleic acid sequences GenBank, EMBL
Protein sequences SWISS-PROT, PIR, PRF, PDBSTR
3D structures PDB
Sequence motifs PROSITE, EPD, TRANSFAC
Enzyme reactions LIGAND
Metabolic pathways PATHWAY
Amino acid mutations PMD
Amino acid indices AAindex
Genetic diseases OMIM
Literature LITDB Medline
Organism-specific gene catalogs E. coli, H. influenzae, M. genitalium, M. pneumoniae, M. jannashii, Synechocystis, S. cerevisiae