Flashcards in Lecture 10 Microbial Genomics Deck (27)
How do we sequence a genome?
-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
1. First Generation sequencing
2. Second generation sequencing
3. Third generation sequencing
What is the Sanger Dideoxy method?
First generation sequencing method
-does PCR with dideoxynucleotides which are analogs
Can do manual
-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?
-Uses Emulsion PCR
-Uses Bridge PCR
What is single molecule sequencing?
Third generation sequencing method
-Real time results, the fastest method
-Does NOT require amplification of DNA samples
What are the methods and platforms of third generation sequencing?
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?
-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?
-140,000 to 1,000,000 bp
-endosymbionts and parasites
-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
-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?
-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
-separate and ID proteins
-predict proteins from DNA
How do genomes change over time?
Horizontal gene transfer
How do we compare genes?
-simlar gene sequences from a common ancestor
-similar genes in two different organisms
-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
What is gene deletion?
loss of a segment of DNA in the genome
-common in endosymbionts and parasites
Dependence on host results in 'useless' genes
What are the mechanisms of genome evolution?
-errors in DNA replication