BioInformatics 2

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BioInformatics (2)
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Physical Mapping - I

• Low resolution
• Megabase-scale
• High resolution
• Kilobase-scale or better
• Methods for low resolution mapping
• Somatic cell hybrids (human and mouse or hamster)
• Fast chromosomal localisation of genes
• Subchromosomal mapping possible
• Fluorescence in situ hybridisation (FISH)
• Chromosome painting
• Fractionation of chromosomes by flow cytometry

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Physical Mapping - II

• Methods for high resolution mapping
• Long-range restriction mapping
• Pulsed-field gel electrophoresis (PFGE)
• Assembly of clone contigs
• The double digest problem
• Ordering fragments from a 2 restriction enzyme
  digest
• Sequence Tagged Sites (STSs)
• Sequence fragments in the genome described
  uniquely
• by a pair of PCR primers
• Usually 200-300 bases
• Very useful as landmarks on the physical map
• Can be mapped to individual clones by FISH
• Assembly of STS-content physical maps

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Physical Mapping - III

• Map units (human genome)
• 1 cM 1 Mb
• 1 cR 30 kb
• 1 centiRay 1 chance of a radiation-induced
  break between 2 markers
• Major information resources
• Stanford Human Genome Center (RH maps)
• http//www-shgc.stanford.edu
• Whitehead/MIT Genome Center (STS content maps)
• http//www-genome.wi.mit.edu/
• Centre dEtude du Polymorphisme Humaine - CEPH
  (YAC maps)
• http//www.cephb.fr/bio/ceph-genethon-map.html

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Physical Mapping - IV

• Conclusions
• The value of physical mapping
• Confirmation of chromosomal location of clones
  and genes
• Correction of genetic map errors
• Correlation to genetic map reveals hotand
  cold regions of recombinational activity on
  chromosomes
• Provides useful information for duplicated
  regions
• High resolution mapping provides the framework
  necessary for high quality sequencing of large
  genomic regions

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System for Assembling Markers (SAM)
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DNA Sequencing

• Ordered clone library
• Sequencing of overlapping clones of known order
  as determined by restriction analysis
• Advantage
• Easy ordering of resulting sequence reads
• Disadvantage
• Detailed mapping is time-consuming
• Shotgun sequencing
• Partial digestion of DNA with a 4-cuter enzyme
• Sequencing of randomly overlapping clones
• Computer-aided assembly of reads
• Advantage
• Speed
• Disadvantage
• High data redundancy due to random sequencing
• Not suitable for large genomes (gt300 Mb)

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Assembly of Sequence Contigs

• The problem
• Semi-automated assembly of a contiguous DNA
  sequence from overlapping gel readings
• Steps
• Base identification
• Trimming of ends
• Vector clipping
• Assembly of fragments
• Major software packages
• SequencherTM from GeneCodes Inc., Ann Arbor,
  Michigan
• Platforms PowerMac, Windows NT
• Up to 70 kb contigs
• The Staden package by Staden et al., MRC,
  Cambridge
• PHRED/PHRAP by Green et al., University of
  Washington, Seattle
• Platforms Unix
• Megabase range contigs
• Mutation detection capabilities

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Quality Control of Sequence DataSource US DOE
Joint Genome Institute

• Goals
• Complete sequence continuity across a target
  region (both within and between clones)
• No more than one gap in 200 kb
• Size of all gaps no larger than 1 of the size of
  the total region
• Allowable gaps include
• regions unclonable/unstable in conventional
  cloning vectors
• repetitive regions
• regions with significant secondary structure or
  abnormally high GC content
• Gap size measured by PCR or restriction digest
  analysis
• Accuracy of finished sequence 1 error in 10,000
  bases
• At least 95 double-strand coverage
• Assembly Verification
• a minimum of three independent restriction
  digests
• reassembly with an independent algorithm
• re-sequencing of random clones

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Submission and Annotation of Sequence Data
Source US DOE Joint Genome Institute

• Size of the starting clone is minimum size of
  submission to public databases
• 95 of the sequence represented on both strands
• all ambiguities resolved or annotated
• missing data from the end of a clone allowed if
  sequence overlap is detected with the adjacent
  clone in the tiling path
• Level of annotation
• all sequences annotated in a largely automated
  fashion
• identification of putative or known genes,
  repetitive elements, EST matches and any other
  useful miscellaneous features
• computationally-derived predictions must be
  indicated as such
• Immediate release of finished annotated sequence
• Global assembly of meta-contigs from previously
  submitted data will be performed periodically

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International Strategy Meeting on Human Genome
SequencingBermuda, 25th-28th February
1996Sponsored by the Wellcome Trust

• Summary of agreed principles
• Primary genomic sequence should be in the public
  domain
• Primary genomic sequence should be rapidly
  released
• Assemblies of greater than 1 Kb should be
  automatically released on a daily basis
• Finished annotated sequence should be immediately
  submitted to the public databases
• Coordination
• Large-scale sequencing centres should inform HUGO
  of their intention to sequence particular regions
  of the human genome

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Annotating the Human Genome Sequence

• Identification of coding regions
• Exon/intron prediction
• High throughput comparison of genomic sequence to
  protein information
• Full-length protein sequences
• Databases of protein domains
• How automated is automated annotation in reality?
• Advantages
• High speed
• Good for tRNA genes, repetitive regions
• Good for high-scoring matches in databases, but
• Disadvantages
• Error propagation can be detrimental
• Domain recycling in evolution causes
  misinterpretation, e.g. in the case of
  transcription factors similar to peptidases
• Very computer-intensive task!