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Flashcards in Lecture 16 Deck (14)

The shotgun approach of whole genome sequencing:

- 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


Joining two contigs to develop contig scaffolds:

- 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


Why do we need a physical map?

- 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)


Comparing genomes using comparative genomics compares:

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


Single copy markers are the most useful:

- We know where they are and what they code for


Genetic vs physical maps:

- The idea map will integrate physical and genetic data


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

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


Concept of allelic series:

- 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.


Micro satellites:

- 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


How do we score micro satellites markers in a species?

- 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


Advantages of micro satellites/SSR markers:

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


Disadvantages of micro satellites/SSR markers:

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


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

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


Markers can be used for:

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