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
What is genomics? Transcriptomics? Proteomics?
Genomics - all genetic information in the cell
-metagenomics all genetic information in the environment
Transcriptomics - expressed genetic information
Proteomics - translated genetic information
What is metagenomics?
pooled DNA from an environmental smaple
-includes genes from many different organisms
What is transcriptomics?
Study of total gene expression
What are microarrays?
Gene chips
- silica chips containing different genes
- tracks expression levels of individual genes
What are the applications of transcriptomics?
study of pathogenic bacteria
study of human cancer cells
What is proteomics?
study of total protein production
In vitro
-separate and ID proteins
In silico
-predict proteins from DNA
How do genomes change over time?
Gene duplication
Gene deletion
Horizontal gene transfer
How do we compare genes?
Homologous genes
-simlar gene sequences from a common ancestor
Orthologs
-similar genes in two different organisms
Paralogs
- similar genes in the same organism
- arise from gene duplication
What is gene duplication?
segment of DNA copied in the genome
- main mechanism of new gene evolution
- one copy remains unchanged and functional
- one copy mutates to a new function
