Lecture 10 Microbial Genomics Flashcards
How do we sequence a genome?
Shotgun approach
- fragmentation = cut genome into small pieces
- sequencing = sequence each DNA piece
- assembly = put the pieces together
What is fragmentation?
First step in sequencing a genome
- cut the genome into small pieces
- through physical shearing or enzymatic methods
What is sequencing?
Second step of genome sequencing
-determining the nucleotide composition of DNA
Sequencing Methods
- First Generation sequencing
- Second generation sequencing
- Third generation sequencing
What is the Sanger Dideoxy method?
First generation sequencing method
-does PCR with dideoxynucleotides which are analogs
Can do manual
- 4 rxns
- 1 for each nucleotide
- separate w/ gel electrophoresis
Can do automated
-single rxn w/ fluorescent markers fr each nucleotide
What is next generation sequencing?
Second generation sequencing method
-massivle parallel=diff sequences run together
Much fast and cheaper that 1st gen
Still requires amplification of DNA samples
What are the limitations of first generation sequencing?
one sequence at a time
multiple not possible
What are the methods and platforms of second generation sequencing?
454 Platform
-Uses Emulsion PCR
Illumina Platform
-Uses Bridge PCR
What is single molecule sequencing?
Third generation sequencing method
- massively parallel
- Real time results, the fastest method
- Does NOT require amplification of DNA samples
What are the methods and platforms of third generation sequencing?
Nanopore Platform
-Nanopore technology
Pacific Biosciences Platform
-Uses SMRT technology
What is assembly?
Last step of genome sequencing
-reconstructing genomes by combining short, overlapping DNA sequences
What are the types of assembly?
Reference alignment
- comparison to known genome
- must be closely related to organism
De novo assembly
- novel genome construction
- no close relative required
What is bioniformatics?
analyzing and storing DNA/Protein sequences using powerful computational tools
-Doing comparative analysis on genome size, content and organization
What is the difference between a small and large genome?
Small genome
- 140,000 to 1,000,000 bp
- endosymbionts and parasites
Large genome
- 5,000,000 to 13,000,000 bp
- free living organisms
How does gene content differ?
Larger genomes = more genes
-little ‘junk’ DNA
Essential Genes for life
- DNA replication
- translation for protein synthesis
What is annotation?
predicting functional genes from DNA sequence data
-uses comparative analysis
