Bioinformatics

1
Bioinformatics

• Basic concepts recommended starting place text
• Alberts, B et al Molecular Biology of the Cell
  (1994 edition available on-line via
  www.ncbi.nlm.nih.gov in the books link)
• Central Dogma DNA -gt RNA -gt Protein
• transcription translation
• DNA genome, chromosomes (histones), genes, gene
  structure (promotors, regulatory elements,
  start/stop codons, open reading frames, introns,
  exons), nucleotide sequence (ACGT)
• RNA mRNA, nucleotide sequence (ACGU), gene
  expression, alternative splicing (2.6 transcripts
  / gene), tRNA, genetic code, redundancy
• Protein proteome, protein complexes, subunits,
  protein structure, motifs and domains, protein
  sequence (20 amino acids), post-translational
  modifications
• And then there are Molecular interactions,
  signal transduction, metabolism And Cells,
  tissues, systems

2
Bioinformatics Demo/Tutorial

• Based on (primarily)
• Wolfsberg, TG et al A users guide to the human
  genome Nature Genetics Suppl Sept 2002
• Day 1 Human Genome, Key Portals, Gene
  information
• Day 2 Single Nucleotide Polymorphisms,
  LocusLink, OMIM, Comparative Genomics
• Day 3 Proteomics, techniques, databases,
  knowledge bases, Protein information

3
Human Genome

• Project basics
• Ref Putting it together
• Users Guide to Genome Nature Genetics Suppl 32
  (2002)
• Genome sequence databases (with analysis tools)
• Associated knowledge bases
• Literature
• Numerous databases (e.g. pathway databases)
• Databases devoted to high throughput experiment
  data
• Gene expression
• SNP analysis
• And as technologies continue to develop
  proteome analysis
• Ref Databases 2003 Nucl Acids Res 31 (1) 2003

4
Human Genome Project

• Update www.ncbi.nlm.nih.gov/genome/seq
• Sequencing
• Technology allows reading 500-800 bases of a
  sequence per run
• Therefore, genome must be sequenced in fragments
  of DNA (500-800bp)
• Sequence one or both ends
• Assemble fragments back into complete genome
  (several strategies)
• The future is now going beyond sequence

5
Public project strategy

• Clone fragments into bacterial artificial
• chromosomes (BAC) subclone good BACs
• Sequence BACs to achieve 4-5X coverage
• Overlapping BACs form longer sequence contigs
• Contigs are assembled and a build is
  constructed
• Entire draft of genome htgs_draft
• 3. When coverage reaches 8-10X it is quality
  sequence
• Smaller of contigs more continuous sequence
  w/o gaps
• Accession number for clone is same but version
  number increases (e.g. AC108475.2 becomes
  AC108475.3)
• Sequence of this quality is labeled htgs_fulltop
• Finisher identifies and fills remaining gaps
• While underway htgs_activefin
• When finished htgs_phase 3 (used for BLAST (Basic
  Local Alignment Search Tool) queries of
  non-redundant sequences)

6
Submit sequence /Assemble genome

• Sequences are submitted in their draft, interim
  and finished forms to any one member of the
  International Sequence Consortium (US, Europe,
  Japan)
• Accession number and key (i.e. htgs_draft, etc.)
  are assigned.
• Data is exchanged between members nightly.
• The assembly process (build)
• Input data for a build includes draft and
  finished sequence.
• BACs are assembled into contigs and contigs are
  assembled onto chromosomes (using e.g. sequence
  tagged sites, annotations, mapping of clone, info
  from sequencing labs)
• Contaminated sequence, repetitive sequence,
  overlaps and redundant clones or contigs are
  removed and gaps are noted.

7
NCBI Reference Sequences (RefSeq)

• www.ncbi.nlm.nih.gov/Locuslink/RefSeq.htm
• Goal Provide the single best non-redundant and
  comprehensive collection of naturally occurring
  biological molecules, representing the central
  dogma reference sequence
• Each alternatively spliced transcript has own
  mRNA/protein
• Sequence entry is automated and most entries in
  RefSeq are provisional. Once entry is reviewed
  and curated (manual) it is labeled reviewed.
• NC_ and NG_ - complete and regional
  (genomic DNA)
• NT_ and NW_ - Intermediate genomic
  assemblies
• NR_ and NM_ - RNA (non-coding) and mRNAs
• NP_ - proteins

8
RefSeq continued

• Model mRNA sequences other GenBank mRNAs
  aligned with genome transcript sequence
  extracted
• Model sequences may differ from reference
  sequences because of real sequence polymorphisms,
  errors in genomic or mRNA sequences or problems
  with alignment.
• XM_ - model mRNAs
• XP_ - model proteins

9
Annotation
Adding sequence features and experimental
data Ref Stein, L. Nature Rev. Genet. 2
493-503 (2001)

• Known genes
• NCBI aligns RefSeq and GenBank mRNAs to
  assembly
• (selects best align if more than one or if same
  both marked)
• Ensembl aligns all known human proteins to
  assembly (SPTREMBL protein database and a
  protein-to-DNA matcher program)
• USCS aligns RefSeq and GenBank mRNAS to
  assembly (uses BLAT (BLAST like alignment tool)
  to make alignments)
• Predicted genes
• All sites offer gene prediction approaches that
  typically use one or more sensors (e.g.
  association with GC rich regions, motifs)
  Comparison in Ref Genome Res 10, 483 (2000)
• Other annotations
• SNPs, sequence tagged sites, expressed sequence
  tags, repetitive elements, clones

10
The three primary portals

• Ensembl
• Comprehensive human genome annotation and
  sequence based search tools (gene prediction and
  putative gene function and expression).
• Map, gene or protein centric views.
• Ensemble system can be downloaded
• UCSC Genome Browser
• Genome viewed at any scale by an intuitive
  overlay of tracks (e.g. tracks known or
  predicted genes, alternative splices)
• Comparative genomics emphasis - alignment w/mouse
  genome
• Access to BLAT algorithm (BLAST Like Alignment
  Tool)
• NCBI
• Hub for genome-related resources maintains
  GenBank
• Map Viewer tool to visualize experimentally
  verified genes, predicted genes, genomic markers,
  physical and genetic maps and sequence variation
  data

11
Demonstration

• Introducing 3 key interfaces (portals)
• NCBI
• USCS
• Ensembl
• Finding a gene of interest, the genes structure,
  nearby genes and retrieving its sequence.
• Reference Users Guide to the Genome Question 1
  and part of Question 6.

12
NCBI Portal

• www.ncbi.nlm.nih.gov
• Background / guide info, Analysis tools and other
  resources (e.g. molecular biology DB) are indexed
  on home page
• Tool bar on top of page, left and right page edge
  columns
• Identifying info about a gene of interest
• handout with steps

13
USCS Portal

• genome.ucsc.edu
• Background / guide info, Analysis tools, Other
  resources (e.g. molecular biology DB)
• Identifying info about a gene of interest
• handout with steps

14
Ensembl Portal

• www.ensembl.org
• Background / guide info, Analysis tools, Other
  resources (e.g. molecular biology DB)
• Identifying info about a gene of interest
• handout with steps

15
Finding Gene Information

• Review Q1 - Do this on your own with ADAM2
  (remember results arent identical) and then with
  a gene you may be interested in.
• Q6 Gene Sequence Retrieval and Gene Structure
  (skipping flanking region primer design)
• Q8 Finding members of gene families and BLAST
• These questions will be put on the class website
  after class.