Lecture 16

Question 1

The shotgun approach of whole genome sequencing:

Answer 1

• Cut many genome copies into random fragments via shearing
• Make a library of clones fragments
• Sequence everything
• ## Assembly reads into contigs (in the correct order) by overlapping the sequence reads

Question 2

Joining two contigs to develop contig scaffolds:

Answer 2

• Paired-end reads may be used to join two contigs, and this closes the gap between sequenced contig reads.
• A long-insert vector with a sequence read 1 and sequence read 2 can bring random contigs into the same spot

Question 3

Why do we need a physical map?

Answer 3

• It allows positional cloning via walking
• It allows bridging gaps in whole genome assembly
• It assesses genome assembly quality
• IT is a source of potential genetic markers and probes (FISH probes in cytology)
• Detailed analysis of particular (usually difficult) regions of the genome (eg. TE, gene duplications and other structural variations)

Question 4

Comparing genomes using comparative genomics compares:

Answer 4

• Size
• karyotpe (number and appearance of chromosomes)
• complexity (copy number, repeats)
• gene content (synteny) (loss/gain, homology etc)
• gene order (colinearity)

Question 5

Single copy markers are the most useful:

Answer 5

• We know where they are and what they code for

Question 6

Genetic vs physical maps:

Answer 6

• The idea map will integrate physical and genetic data

Question 7

Anchor loci can help reeal chromosomal conservation or rearrangements. Their properties include:

Answer 7

• Single copy
• Highly conserved (can compare closely related species)
• Unique (in terms of insertions, deletions, etc)

Question 8

Concept of allelic series:

Answer 8

• There are many ways to alter a gene sequence
• A gene can have many alleles in the natural populations (it is not just mutant and WT)
• This is called an allelic series and there is an order of dominance.

Question 9

Micro satellites:

Answer 9

• Simple sequence repeats (SSRs) or short tandem repeats (STRs)
• There can be a difference in the number of repeat units, but they have conserved left and right flanking sequences
• These could arise via replication slippage

Question 10

How do we score micro satellites markers in a species?

Answer 10

• Extract DNA and cut it.
• Clone fragments
• Grow clones in bacteria and hybridise with a SSR probe
• Sequence positive clones to confirm SSR and obtain flanking sequences
• Design primers flanking SSR
• Design primers based on known flanking sequences
• Purify the DNA and go through PCR amplification and electrophoresis
• Differenc mircrosatellites can be observed

Question 11

Advantages of micro satellites/SSR markers:

Answer 11

• Very polymorphic
• Usually abundant
• Co-dominant
• Only requires small amounts of DNA
• Same method can be used for all species
• Selectively neutral

Question 12

Disadvantages of micro satellites/SSR markers:

Answer 12

• Library construction can be costly and time consuming
• Per phenotype cost high compared to NSG-based techniques
• Is it really neutral?

Question 13

We can connect two species via their genetic map using micro satellites:

Answer 13

This allows a web of relationships between physical maps and genetic maps from two species
- This is the primary use of microsatellites

Question 14

Markers can be used for:

Answer 14

• Gene flow
• Conservation
• Linkage mapping
• QTL mapping
• Genetic counselling
• Paternity testing
• Pathogen identification
• GWAS
• Evolution