BioInformatics (2) - PowerPoint PPT Presentation

• 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

• 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

• 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 d’Etude du Polymorphisme Humaine - CEPH (YAC maps)
• http://www.cephb.fr/bio/ceph-genethon-map.html

• Conclusions
• The value of physical mapping
• Confirmation of chromosomal location of clones and genes
• Correction of genetic map errors
• Correlation to genetic map reveals ‘hot’and ‘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

System for Assembling Markers (SAM)

• 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 (>300 Mb)

• 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

• 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

• 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

• 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

• 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!