Download Molecular markers

Molecular markers
What are molecular markers?
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A readily detectable sequence of DNA or a
protein whose inheritance can be
monitored
Polymorphism in proteins
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Allozymes and isozymes
Polymorphism in DNA
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Nuclear
Cytoplasmic
Desirable properties of molecular markers
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Polymorphic
Codominat inheritance
Easy, fast and inexpensive to detect
Reproducible, transferable
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No single marker meets all needs!
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Isozymes and allozymes
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Around 1960
Multiple forms of the same enzyme
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Isozyme:one enzyme, more than one gene
locus
Allozyme:one enzyme, one gene locus
Methodology
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Macerate tissue
Separate enzymes by electrophoresis
Locate enzymes by histochemical staining
Analyze banding patterns
Allozymes:methodology
Enzyme electrophoresis:
Advantages
n Robust, cheap
reproducible method
for:
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Characterizing/
identifying genotypes
Studying population
genetics
Examining geographical
patterns of variation
Disadvantages
n Limited number of
enzymes available
n Limited amount of
variation
RFLP
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Restriction Fragment Length Polymorphism
Around 1970
RFLP examines differences in size of
specific DNA restriction fragments
Requires pure, high molecular weight DNA
RFLP: methodology
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Cut DNA into smaller fragments
Separate fragments by gel
Transfer DNA fragments to a filter
Visualise DNA fragments
n radioactive probes
n non-radioactive probes
RFLP: analysis of results
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Bands scored for presence/absence
Differences reflect genetic differences
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the choice of restriction enzyme crucial
RFLP
Advantages
n Reproducible
n Co-dominant markers
n Simple
Disadvantages
n Time consuming
n Expensive
n Use of radioactive
probes
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not needed for
mitochondrial/
chloroplast DNA
Minisatellites
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Invented in 1985 (Jeffreys et al.)
Like RFLP, but the probe binds in
microsatellite area (tandem repeats, 10-50
base pairs)
Multi locus minisatellites (DNA-fingerprints)
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paternity analysis, not very useful for
population analysis
Single locus minisatellites (VNTR = Variable
number of tandem repeats)
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more useful for population analysis
Minisatellites: interpretation
RAPD
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Random Amplified Polymorphic DNA
1990 (Welsh et al.)
PCR-based method
Procedure: amplify anonymous stretches of
DNA by using arbitrary primers
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one primer/reaction
generally 10 bases (depends on genome size)
Separate fragments by agarose gel
Locate fragments by total DNA-staining
RAPD: interpreation
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Bands scored for presence/absence
RAPD
Advantages
n Fast and simple
n Inexpensive
n No radioisotopes
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Disadvantages
Dominant markers
Reproducibility
problems
Problems of
interpretation
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Same band, same
fragment
Microsatellites
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1989 (Tautz, Litt & Luty) (originally CA)
Sequence tagged microsatellites (STMS) or
Simple Sequence Repeat Polymorphism (SSRP) or
Short Tandem Repeats (STR)
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Motif (1-6 base pairs) repeats e.g.
CGCGCGCGCGCGCGCG
Procedure: amplify with PCR with specific primers
Separate fragments by polyacryalamide
sequencing gel (+ stain with silver nitrate) (or
agarose + total DNA-stain) or use automated
DNA detection with fluorescent based technology
Microsatellites
Microsatellites
Advantages
n High amount of
variation
n Codominant
n Highly reproducible
n More useful than
minisatellites
n Known mutation
model (?)
Disadvantages
n Development difficult
(separate sheet) and
expensive
n Interpretation
sometimes difficult
(stutter bands)
Microsatellites: examples
Microsatellites: examples
AFLP
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1995 (Vos et al.)
Amplified Fragment Length Polymorphism
AFLP is a DNA fingerprinting technique
which detects DNA restriction fragments by
means of PCR amplification.
AFLP: technology
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Comprises of the following steps:
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The restriction of the DNA with two restriction
enzymes, preferably a hexa-cutter and a tetracutter
The ligation of double-stranded (ds) adapters
to the ends of the restriction fragments
The amplification of a subset of the restriction
fragments using two primers complementary to
the adapter and restriction site sequences, and
extended at their 3' ends by "selective
nucleotides"
AFLP: methodology
AFLP: methodology
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Gel electrophoresis of the amplified
restriction fragments on denaturing
polyacrylamide gels ("sequence gels");
The visualization of the DNA fingerprints by
means of autoradiography, phosphoimaging, or other methods.
AFLP: example
AFLP
Advantages
n No prior gene or
sequence information
is required
n More reliable than
RAPD
n The technique is very
sensitive, and is
consequently able to
detect low abundance
transcripts
Disadvantages
n Mostly dominat
n Technically demanding
and expensive
Amplifying a specific loci
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Single nucleotide polymorphism (SNP)
Disadvantages
Advantages
n Formerly labour-intensive to
n It is known what is
amplified: known source discover sequences
of polymorphism
n Formerly rely on relatively few
loci: violation of assumptions
n Often more reliable than
Biallelic
anonymous methods
(specific primers)
n Not known mutation model
Kwok P. 2001. METHODS FOR GENOTYPING SINGLE NUCLEOTIDE
POLYMORPHISMS. Annual Review of Genomics and Human Genetics
2: 235-258; DOI: 10.1146/annurev.genom.2.1.235
SNP
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SNP discovery by alignment of sequence traces
obtained from direct sequencing of genomic PCR products.
SNP-revolution
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SNP arrays available for
model species
NGS-sequencing allows
easy detection of SNP:s
even for non-model
species
Next de facto markers for
population genetic
CanineHD BeadChip
•More than 170,000 evenly
studies?
spaced and validated SNPs
Genomic level markers
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With the advent of next-generation sequencing (NGS),
there are several approaches, which are capable of
discovering, sequencing and genotyping not hundreds
but thousands of markers across almost any genome of
interest in a single step, even in populations in which
little or no genetic information is available.
Kumar et al. 2012. SNP Discovery through NextGeneration Sequencing. -Int. J. Plant Genom.
(http://www.hindawi.com/journals/ijpg/2012/831460/)
Davey et al. 2011. Genome-wide genetic marker
discovery and genotyping using next-generation
sequencing. –Nature Reviews Genetics 12: 503
Platforms
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First Generation Sequencing
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(long reads, high quality, low throughput, high cost)
Sanger Sequencing
Second Generation Sequencing (PCR Needed) decreased
cost, short reads,
high throughput (massively parallel), clonal template amplification
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Third Generation Sequencing (Single Molecule Sequencing )
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Roche: 454 (pyrosequencing) X
Life Science: IONtorrent & IONproton
Illumina: MiSeq & HiSeq (Sequencing by Synthesis)
ABI: SOLiD
Helicos Biosciences: true single molecule sequencing (tSMS)
Pacific Biosciences: PacBio RS II (Non--terminal fluorescent sequencing
by synthesis, SMRT)
Oxford Nanopore: MinION & GridION (single nucleotides are detected as
they pass through a nanopore)
http://www.molecularecologist.com/next-gen-fieldguide-2014/
Dideoxy sequencing (Sanger)
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1977
Selective termination
of DNA-synthesis
Four reactions (lanes)
Separate by
denaturing PAGE
Visualize by
autoradiography
Automatic Sanger sequencing
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Fluorescent dyes: one lane, four bases
SGS-methods: Pyrosequencing 1998
Ronaghi M., Uhlen M., Nyren P. (1998b) A sequencing method based on real-time pyrophosphate.
Science 281:363–365
Ion Torrent
Based on monitoring of base incorporation during DNA synthesis,
similar to 454
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1. Beads placed in wells of a semiconductor chip which contain a pH
sensing layer
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2. Single nucleotide types are flowed across the chip one at a time
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3. Incorporation of a nucleotide by DNA polymerase results in the
release of a hydrogen ion, which is detected and recorded.
– Multiple incorporations lead to a larger signal
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Illumina sequencing
Metzker 2010
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https://www.youtube.com/watch?v=HMyCqWhwB8E
Illumina sequencing
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Also based on observation of incorporated nucleotides
during DNA synthesis
Clonal amplification of template DNA on a glass flow
cell
Fluorescent, reversibly-terminated nucleotides are added
with DNA polymerase.
‘Stalled’ DNA synthesis occurs because of terminating
nucleotides
System is photographed/scanned in A, G, C and T
channel
Terminator and fluorophore are removed
TGS: Oxford nanopore
Oxford nanopore
SNP-discovery
SNP-calling
SNP-validation
SNP-genotyping
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Kumar et al. 2012. SNP Discovery through
Next-Generation Sequencing. -Int. J. Plant
Genom.
SNP-discovery
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SNP is identified when a nucleotide from an
accession read differs from the reference
genome at the same nucleotide position.
Graphical user interface of Tablet, an assembly visualization program, displays the
reference genome on top and the mapped reads with color-coded SNPs on the bottom.
SNP-discovery
Complexity reduction strategies
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Often use restriction enzyme digestion of target
genomes to reduce the complexity
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Reduced-representation sequencing using reducedrepresentation libraries (RRLs)
Complexity reduction of polymorphic sequences
(CRoPS),
restriction-site-associated DNA sequencing (RAD-seq)
low coverage genotyping including MSG and
genotyping by sequencing (GBS)
Davey et al. 2011. Genome-wide genetic marker discovery and
genotyping using next-generation sequencing. –Nature Reviews Genetics
12: 503
Genotyping by sequencing (GBS;
right panels):
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barcoded adaptors (yellow) and
common adaptors (grey) are ligated
to digested fragments, producing
fragments with barcode+common,
barcode+barcode and
common+common adaptor
combinations.
Samples are pooled and amplified
on the Illumina Genome Analyzer
flowcell.
Only short samples featuring a
barcode+common adaptor
combination are amplified for
sequencing
Cost of sequencing