Transcript PPT - Blumberg lab

BioSci 145B Lecture #2 4/13/2004
• Bruce Blumberg
– 2113E McGaugh Hall - office hours Wed 12-1 PM (or by appointment)
– phone 824-8573
– [email protected]
• TA – Curtis Daly
– 2113 McGaugh Hall, 924-6873, 3116
• lectures will be posted on web pages after lecture
– http://eee.uci.edu/04s/05705/ - link only here
– http://blumberg-serv.bio.uci.edu/bio145b-sp2004
– http://blumberg.bio.uci.edu/bio145b-sp2004
• On 3 x 5 card write
– On lined side – something that you recall clearly from last week’s class
– On other side – something that was unclear to you from last week
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Requirements for the oral presentation
• Goal – again to get you to think more analytically
– Exposure to literature (classic and current)
– Learn critical reading
– Discuss practical applications of what we are learning
• Powerpoint (“journal club”) presentation – as a presenter
– 20 minutes with time allowed for discussion (max of 15 – 20 slides)
– Frame the problem – what is the big picture question?
• What was known before they started? What was unknown?
• Present background (few slides), handouts helpful but not required
– What are specific questions or hypotheses to be tested
• Discuss figures
– What is the question being asked in each figure or panel?
– What experiments did the authors do to answer questions?
– Do the data support the conclusions drawn?
» Were controls done?
• What did they conclude overall?
• What could have been improved?
– Point out a few papers for further reading (reviews, followups, etc)
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Requirements for the oral presentation (contd)
• Powerpoint presentation – as a listener
– READ THE PAPERS
– Study the figures
• What points don’t you understand?
– Make notations, ask the speaker to clarify these
– Listen to the speaker
• If presentation is unclear, ask the speaker to elaborate
• Always feel free to ask questions – we want an open discussion
• Papers are posted on the web sites listed
– I have CDs for everyone
• Logistics
– Prepare presentation and either e-mail to me or bring it on a
• CD-ROM
• Floppy disk
• USB flash memory drive
– Or bring your own laptop
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Presentation schedule
•
•
•
Week 1 – Berget et al., 1977; Chow et al., 1977 - Curtis
Week 2 – (1) Geisler et al., 1999 (2) Maniatis et al., 1978 (3) Osoegawa et al., 2000
– Erik
Week 3 – (4) Adams et al., 1991 (5) Sargent and Dawid, 1983 (6) Wang and Brown, 1991
•
Week 4 - (7) Innis et al., 1988 (8) Myers et al., 2000 (9) Sanger et al., 1977
•
Week 5 – midterm, no presentations
•
Week 6 –(10) Hsu et al., 2002 (11) Tyson et al., 2004 (12) Venter et al., 2004
•
Week 7 – (13) Cawley et al., 2004 (14) Kapranov et al., 2002 (15) Schena et al., 1996
•
Week 8 – (16) Boutros et al., 2004 (17) Carlson et al., 2003 (18) Golling et al., 2002
•
Week 9 – (19) Fields and Song, 1989 (20) Ito et al., 2001 (21) Uetz et al., 2000
•
Week 10 - (22) Gavin et al., 2002 (23) Hamadeh et al., 2002 (24) Hoffmeyer et al.,
2000
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Lecture Outline 4/13/2004 – Mapping Genomes
• Today’s topics
– Finish up from last time
– Construction of genomic libraries
– Modern mapping methods
• The big picture – How does one go about creating a genomic map?
– In preparation for sequencing
– To locate disease loci
• This week’s papers – Classic paper on library construction and screening and
two recent ones, one about library construction the other about radiation
hybrid mapping
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Origins of intron/exon organization
• When did introns arise
– Introns early – Walter Gilbert
• There from the beginning, lost in bacteria and many simpler
organisms
– Introns late – Cavalier-Smith, Ford Doolittle, Russell Doolittle
• Introns acquired over time as a result of transposable elements,
aberrant splicing, etc
• If introns benefit protein evolution – why would they be lost?
– Which is it?
• Introns late
(at the moment)
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Evolution of gene clusters
• Many genes occur as multigene families (e.g., actin, tubulin, globins, Hox)
– Inference is that they evolved from a common ancestor
– Families can be
• clustered - nearby on chromosomes (α-globins, HoxA)
• Dispersed – on various chromosomes (actin, tubulin)
• Both – related clusters on different chromosomes (α,β-globins,
HoxA,B,C,D)
– Members of clusters may show stage or
tissue-specific expression
• Implies means for coregulation as well
as individual regulation
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Evolution of gene clusters (contd)
• multigene families (contd)
– Gene number tends to increase with
evolutionary complexity
• Globin genes increase in number from
primitive fish to humans
– Clusters evolve by duplication and divergence
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Evolution of gene clusters (contd)
• History of gene families can be traced by comparing sequences
– Molecular clock model holds that rate of change within a group is
relatively constant
• Not totally accurate – check rat genome sequence paper
– Distance between related sequences combined with clock leads to
inference about when duplication took place
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Types and origin of repetitive elements
• DNA sequences are not random
– genes, restriction sites, methylation sites
• Repeated sequences are not random either
– Some occur as tandemly repeated sequences
– Usually generated by unequal crossing
over during meiosis
– These resolve in ultracentrifuge into
satellite bands because GC content
differs from majority of DNA
– This “satellite” DNA is highly variable
• Between species
• And among individuals within a
population
• Can be useful for mapping
genotyping, etc
– Much highly repetitive DNA is in
heterochromatin (highly condensed regions)
• Centromeres, telomeres, others
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Types and origin of repetitive elements (contd)
• Dispersed tandem repeats are “minisatellites” 14-500 bp in length
– First forensic DNA typing used satellite DNA – Sir Alec Jeffreys
– Minisatellite DNA is highly variable and perfect for fingerprinting
BioSci 145B lecture 2
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Bruce Blumberg 2004. All rights reserved
Types and origin of repetitive elements – dispersed repeated sequences
BioSci 145B lecture 2
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Bruce Blumberg 2004. All rights reserved
Types and origin of repetitive elements – dispersed repeated sequences
• Book discusses types of elements, similarities, differences at great length
– Main point is to understand how such elements can affect evolution of
genes and genomes
– Gene transduction has long been known in bacteria (transposons, P1, etc)
– LINE (long interspersed nuclear elements)
can mediate movement of exons between
genes
• Pick up exons due to weak polyadenylation signals
• The new exon becomes part of LINE
by reverse transcription and is
inserted into a new gene along
with LINE
– Voila – gene has a new exon
– Experiments in cell culture proved this
model and suggested it is
unexpectedly efficient
– Likely to be a very important mechanism
for generating new genes
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome Structure
• The big picture
– Chromosomes consist of coding (euchromatin) and noncoding
(heterochromatin) regions
• Various physical methods can distinguish these regions
– Staining
– Buoyant density
– Restriction digestion
• Heterochromatin is primarily tandemly repeated sequences
• Euchromatin is everything else
– Genes including promoters, introns, exons
– LINES, SINES micro and minisatellite DNA
• Patterns of euchromatin and heterochromatin can be useful for
constructing genetic maps
– Heterochromatin is trouble for large scale physical mapping and
sequencing
• May be hard to cross gaps
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome evolution
• Genomes evolve increasing complexity in various ways
– Whole genome duplications
• Particularly important in plants
– Recombination and duplication mediated by SINEs, LINEs, etc.
• Expands repeats, exon shuffling, creates new genes
– Meiotic crossing over
• Expands repeats, duplicates genes
– Segmental duplication – frequent in genetic diseases
• Interchromosomal – duplications among non-homologous
chromosomes
• Intrachromosomal – within or across homologous chromosomes
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome evolution (contd)
• Several recent papers discuss details
of genome evolution as studied in
closely related species
– Dietrich et al. (2004)
Science 304, 304-7
– Kellis et al. (2004)
Nature 428, 617-24.
– S. cerevvisiae vs two other
species of yeast
• Saw genome duplications
and
• evolution or loss of
one duplicated member
but never both
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Mapping Genomes
• Why map genomes?
– Locate genes causing mutations or diseases
• Figure out where identified genes are
– Prepare to sequence
– Discern evolutionary relationships
• How do we go about mapping whole genomes?
– Book describes restriction endonuclease digestion
• Impossible for all but the tiniest genomes
• Requires ability to precisely resolve very large fragments of DNA
– Must be able to separate chromosomes or huge fragments thereof
• Then map various types of markers onto these fragments
• STS, ESTs, RFLPs
– Modern approach
• Construct large insert genomic libraries
– Map relationship to each other
– map markers onto large insert library members
• Map to chromosomes
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Construction of genomic libraries
• What do we commonly use genomic libraries for?
– Genome sequencing
– gene cloning prior to targeted disruption or promoter analysis
– positional cloning
• genetic mapping
– Radiation hybrid, STS (sequence tagged sites), ESTs, RFLPs
• chromosome walking
• gene identification from large insert clones
• disease locus isolation and characterization
• Considerations before making a genomic library
– what will you use it for
• what size inserts are required?
– Are high quality validated libraries available?
• Caveat emptor
– Research Genetics X. tropicalis BAC library is really Xenopus
laevis
• apply stringent standards, your time is valuable
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genomic libraries (contd.)
• Considerations before making a genomic library (contd)
– availability of equipment?
• PFGE
• laboratory automation
• if not available locally
it may be better to use
a commercial library
or contract out the
construction
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genomic libraries (contd.)
• Goals for a genomic library
– Faithful representation of genome
• clonability and stability of fragments essential
• >5 fold coverage i.e., library should have a complexity of five times
the genome size for a 99% probability of a clone being present.
– easy to screen
• plaques much easier to deal with colonies UNLESS you are dealing
with libraries spotted in high density on filter supports
– easy to produce quantities of DNA for further analysis
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Construction of genomic libraries
• Prepare HMW DNA
– bacteriophage λ, cosmids or fosmids
• partial digest with frequent (4) cutter followed by sucrose gradient
fractionation or gel electrophoresis
– Sau3A (^GATC) most frequently used, compatible with BamHI
(G^GATCC)
• why can’t we use rare cutters?
• Ligate to phage or cosmid arms then package in vitro
– Stratagene >>> better than competition
– Vectors that accept larger inserts
• prepare DNA by enzyme digestion in agarose blocks
– why?
• Partial digest with frequent cutter
• Separate size range of interest by PFGE (pulsed field gel
electrophoresis)
• ligate to vector and transform by electroporation
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Construction of genomic libraries (contd)
• What is the potential flaw for all these methods?
– Unequal representation of restriction sites, even 4 cutters in genome
– large regions may exist devoid of any restriction sites
• tend not to be in genes
• Solution?
– Shear DNA or cut with several 4 cutters, then methylate and attach
linkers for cloning
– benefits
• should get accurate representation of genome
• can select restriction sites for particular vector (i.e., not limited to
BamHI)
– pitfalls
• quality of methylases
• more steps
• potential for artefactual ligation of fragments
– molar excess of linkers
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Construction of genomic libraries (contd)
• What sorts of vectors are useful for genomic libraries?
– Plasmids?
– Bacteriophages?
– Others?
• Standard plasmids nearly useless
• Bacteriophage lamba once most useful and popular
– Size limited to 20 kb
• Lambda variants allow larger inserts – 40 kb
– Cosmids
– Fosmids
• Bacteriophage P1 – 90 kb
• YACs – yeast artificial chromosomes - megabases
• New vectors BACs and PACs - 300 kb
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Bacteriophage library cloning systems
• All are relatively similar to each other
– Lambda, cosmid, fosmid, P1
– Erik will tell us about lambda on
Thursday
• P1 cloning systems
– derived from bacteriophage P1
• one of the primary
tools of E. coli
geneticists for
many years
– infect cells with packaged
DNA then recover as a
plasmid.
– useful, but size limited
to 95 kb by “headfull”
packaging mechanism
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Cosmid/fosmid cloning
• P1, cosmids and fosmids replicated
as plasmids after infection
– Cosmids have ColE1 origin (2550 copies/cell_
– Fosmids have F1 origin (1
copy/cell)
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Screening a bacteriophage library
BioSci 145B lecture 2
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Bruce Blumberg 2004. All rights reserved
YACs, BACs and PACs
• Three complementary approaches, each with its own strengths and
weaknesses
• YACs - Yeast artificial chromosomes
– requires two vector arms, one
with an ARS one with a
centromere
• both fragments have
selective markers
– trp and ura are
commonly used
• background reduction
is by dephosphorylation
• ligation is transformed
into spheroplasts
• colonies picked into
microtiter dishes containing
media with cryoprotectant
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
YAC cloning
• YAC cloning (contd)
– advantages
• can propagate extremely large fragments
• may propagate sequences unclonable in E. coli
– disadvantages
• tedious to purify away from yeast chromosomes by PFGE
• grow slowly
• insert instability
• generally difficult to handle
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
BAC cloning
• Based on the E. coli F’ plasmid
– partial digests are cloned into dephosphorylated vector
– ligation is transformed into E. coli by electroporation
– advantages
• large plasmids - handle with usual methods
• Stable - stringently controlled at 1 copy/cell
• Vectors are small ~7 kb
– – good for shotgun cloning strategies
– disadvantages
• low yield
• no selection against
nonrecombinant clones
(blue/white only)
• apparent size limitation
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
PAC cloning
• PAC - P1 artificial chromosome
– combines best features of P1 and BAC cloning
– size selected partial digests
are ligated to dephosphorylated
vector and electrotransformed
into E. coli.
• Stored as colonies in
microtiter plates
– Selection against
non-recombinants via
SacBII selection
(nonrecombinant
cells convert sucrose into a
toxic product)
– inducible P1 lytic replicon
allows amplification of
plasmid copy number
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
PAC cloning (contd)
• PAC
– advantages
• all the advantages of BACS
– stability
– replication as plasmids
– stringent copy control
• selection against nonrecombinant clones
• inducible P1 lytic replicon
– addition of IPTG causes loss of copy control and larger yields
– disadvantages
• effective size limitation (~300 kb)
• Vector is large – lots of vector fragments from shotgun cloning PACs
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Comparison of cloning systems
YAC
BAC
PAC
Host cells
S. cerevisiae
AB1380, J57D
E. coli DH10B
E. coli DH10B
Transformation
method
Spheroplast
transformation
Electroporation
Electroporation
DNA topology of
recombinants
Linear
Circular supercoiled
Circular supercoiled
Maximum insert
size
>>1 Mb
~300 kb
~300 kb
Selection for
recombinants
Ade2 supF red-white
color selection
Lacz blue-white
SacIIb selective
growth
Selection for vector
Dropout medium
(lacking trp and ura)
Chloramphenicol
Kanamycin
Enzyme for partial
digests
EcoRI
HindIII
MboI or Sau3AI
Stability
Variable but can be
very unstable
Very stable
Very stable
Degree of
chimerism
Varies but can be
>50%
Very low
Very low
Degree of cocloning
Occasional
Undetectable
Undetectable
Purification of
intact inserts
Difficult
Easy
Easy
Direct sequencing
of insert
Difficult
Relatively easy
Relatively easy
Clone mating
Yes
No
No’
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Which type of library to make
• Do I need to make a new library at all?
– Is the library I need available? http://bacpac.chori.org/home.htm
• PAC libraries are suitable for most purposes
• BAC libraries are most widely available
• If your organism only has YAC libraries available you may wish to
make PAC or BACs
• Much easier to buy pools or gridded libraries for screening
– doesn’t always work
– What is the intended use?
• Will this library be used many times?
– e.g. for isolation of clones for knockouts
– if so, it pays to do it right
– who should make the library?
• Going rate for custom PAC or BAC library is 50K. Most labs do not
have these resources
• if care is taken, construction is not so difficult
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping
• The problem – genomes are large, workable fragments are small
– How to figure out where everything is?
– How to track mutations in families or lineages?
• Book makes a good analogy to roadmaps
– The most useful maps do not have too much detail but have major
features and landmarks that everything can be related to
• Allows genetic markers to be related to physical markers
• What sorts of maps are useful for genomes?
– Restriction maps of various sorts
• RFLPs, fingerprints
– Recombination maps, how often to traits segregate together
– Physical maps – which genes occur on same chunks of DNA
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• How are maps made?
– Restriction digestion and ordering of fragments to build contigs
• Fingerprinting
– Location of marker sequences onto larger chunks
– Hybridization of markers to larger chunks
– Calculation of recombination frequencies between loci
• What do we map these days?
– BACs are most common target for mapping of new genomes
– Radiation hybrid panels still in wide use
– Goal is always to map markers onto ordered large fragments and infer
location of genes relative to each other.
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Useful markers
– STS – sequence tagged sites
• Short randomly acquired sequences
• PCRing sequences, then prove by
hybridization that only a
single sequence is amplified/genome
– VERY tedious and slow
• validated ones mapped back
to RH panels
• Orders sequences on large chunks of DNA
– STC – sequence tagged connectors
• Array BAC libraries to 15x
coverage of genome
• Sequence BAC ends
• Combine with genomic maps
and fingerprints to link clones
– Average about 1 tag/5 kb
• Most useful preparatory
to sequencing
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Useful markers (contd)
– ESTs – expressed sequence tags
• randomly acquired cDNA sequences
• Lots of value in ESTs (paper next week)
– Info about diversity of genes expressed
– Quick way to get expressed genes
• Better than STS because ESTs are expressed genes
• Can be mapped to
– chromosomes by FISH
– RH panels
– BAC contigs
– Polymorphic STS – STS with variable lengths
• Often due to microsatellite differences
• Useful for determining relationships
• Also widely used for forensic analysis
– OJ, Kobe, etc
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Useful markers (contd)
– SNPs – single nucleotide polymorphisms
• Extraordinarily useful - ~1/1000 bp in humans
• Much of the differences among us are in SNPs
• SNPs that change restriction sites cause RFLPs (restriction fragment
length polymorphisms
• Detected in various ways
– Hybridization to high density arrays (Affymetrix)
– Sequencing
– Denaturing electrophoresis or HPLC
– Invasive cleavage
• Tony Long in E&E Biology has new method for ligation mediated SNP
detection that they use for evolutionary analyses
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Useful markers (contd)
– RAPDs – randomly amplified polymorphic DNA
• Amplify genomic DNA with short, arbitrary primers
• Some fraction will amplify fragments that differ among individuals
• These can be mapped like STS
• Issues with PCR amplification
• Benefit – no sequence information required for target
– AFLPs – amplified fragment length polymorphisms
• Cut with enzymes (6 and 4 cutter) that yield a variety of small
fragments ( < 1 kb)
• Ligate sequences to ends and amplify by PCR
• Generates a fingerprint
– Controlled by how frequently enzymes cut
• Often correspond to unique regions of genome
– Can be mapped
• Benefit – no sequence required.
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Mapping by walking/hybridization
– Start with a seed clone then walk along the chromosome
– Takes a LOOONNNNGGG time
– Benefit – can easily jump repetitive sequences
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Fingerprinting
– Array and spot ibraries
– Probe with short oligos (10-mers)
• Repeat
– Build up a “fingerprint” for each clone
– Can tell which ones share sequences
• tedious
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Mapping by hybridization
– Array library – pick a “seed clone”
– See where it hybridizes, pick new seed and repeat
– Product
• Mapping by restriction digest
fingerprinting
– Order clones by comparing
patterns from restriction enzyme
digestion
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• FISH - Fluorescent in situ hybridization – can detect chromosomes or genes
– Can localize probes to chromosomes and
– Relationship of markers to each other
– Requires much knowledge of genome being mapped
– Chromosome painting
BioSci 145B lecture 2
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©copyright
marker detection
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• Radiation hybrid mapping
– Old but very useful technique
• Blast cells with x-rays
• Fuse with cells of another species, e.g., blast human cells then fuse
with mouse cells
– Chunks of human DNA will remain in mouse cells
• Can build up a library of different cell lines – RH panel
– Now map markers onto these RH panels
• Can identify which of any type of markers map together
– STS, EST (very commonly used), etc
• Can then map others by linkage to the ones you have mapped
– We will discuss a paper on Thursday
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved
Genome mapping (contd)
• How should maps be made with current knowledge?
– All methods have strengths and weaknesses – must integrate data for
useful map
• e.g, RH panel, BAC maps, STS, ESTs
– Size and complexity of genome is important
• More complex genomes require more markers and time mapping
– Breakpoints and markers are mapped relative to each other
– Maps need to be defined by markers (cities, lakes, roads in analogy)
– Key part of making a finely detailed map is construction of genomic
libraries and cell lines for common use
• Efforts by many groups increase resolution and utility of maps
• Current strategies
– BAC end sequencing
– Whole genome shotgun sequencing
– EST sequencing
– Mapping of above to RH panels
BioSci 145B lecture 2
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©copyright
Bruce Blumberg 2004. All rights reserved