Lecture 16 Flashcards
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
- GWAS
- Evolution
