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BIO 357 - Microbiology > Microbial Genomics > Flashcards

Microbial Genomics Flashcards

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Biology 101 flashcards
1
Q

Genome

A

entire complement of genetic info including genes, regulatory sequences and noncoding DNA

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2
Q

Genomics

A

discipline of mapping, sequencing, analyzing and comparing genes

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3
Q

Sanger Dideoxy Sequencing Method

A
  • uses radioactively labeled bases in PCR
  • uses gel electrophoresis to sequence the DNA
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4
Q

Illumina Sequencing Method

A
  • 200 million reads/run; 300 bp length
  • Steps:
    • put on double stranded adapters
    • have complimentary strands on flowell (600 million clusters)
    • creates build up/bridges on flowell
      • bridge amplification, stationary phase PCR
    • clusters are identified w/ fluorescence
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5
Q

PacBio Sequencing

A

capable of 30,000 reads/run; 2000 bp length

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6
Q

454 Sequencing

A
  • 1 million reads/run; 700 bp length
  • generates data 100x faster than Sanger method
  • relies on 2 major advances
    • use of robotics
    • pyrosequencing
  • Steps:
    • chop up DNA, attach things w/ ligase, undergo emulsion PCR which attaches DNA to bead
    • everything placed in picotiter plate (1.7 million wells)
    • nucleotides are run over DNA and will attach
      • releases PPi which get transferred to ATP which then admits light
    • light identifiable for each base
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7
Q

How do you assemble the information from sequencing into a genome?

A
  • align all reads w/ software to generate longer sequencing (contigs)
  • gaps remain btwn contigs when don’t match up = “draft” genomes
    • orientation can be assumed if know related organisms
    • can amplify gaps by sequencing PCR amplicons
  • most laborous and expensive part of sequencing
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8
Q

Annotation

A
  • coverting raw sequence data into a list of genes present in genome
    • majority of genes encode mRNAs (open reading frames)
    • some encode functional RNAs (rRNAs, tRNAs, ncRNAs)
  • Functional ORF = Open Reading Frame (encodes proteins)
    • computers search for start/stop codons and shine-dalgarno sequences
      • identify ORF
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9
Q

What is the correlation of gene size to genome size?

A
  • gene = 1,000 bp
  • 1,000 genes = 1 genome
  • 1 Mbp = 1,000 genes = 1,000,000 bp
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10
Q

What is the fractino of noncoding DNA in microbes?

A
  • Eukaryotic microbes = >80%
  • Bacteria = <15%
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11
Q

How many kbp does Nanoarcheaum equitans have?

A

139 kbp

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12
Q

How many kbp does *Mucobacterium tuberculosis *have?

A

4,400 kbp

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13
Q

What is the estimated minimum # of genes needed for cell viability?

A

250 - 400 genes

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14
Q

Which prokaryote has the larges genome to date at 13.0 Mbp?

A

Sorangium cellulosm

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15
Q

Do archaeal genomes tend to be smaller or larger than bacterial ones?

A

smaller at only an estimated ~5 Mbp

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16
Q

What is the general gene distribution in prokaryotes?

A
  • the most abundant class is metabolic genes
  • a minor class is made up of DNA replication and transcription genes
  • prevalent class is those genes for nontranslated RNAs (rRNA, tRNA, ncRNA)
17
Q

Compare gene distribution between Bacteria and Archaea:

A
  • archaea devote higher % to energy and coenzyme production
  • bacteria devote higher % to carbohydrate metabolism and cell membrane fxn
18
Q

Metagenome

A
  • total gene content of the organisms present in an environment
  • usually used to analyze all of 16S genes, and then compare to determine present organisms
19
Q

Transcriptome

A
  • the entire complement of RNA produced under a given set of conditions
20
Q

Why are Transcriptomes studied?

A
  • to analyze …
    • global gene expression
    • expression of specific groups of genes under diff conditions
    • expression of genes w/ unknown fxn; can yield clues to its role
    • comparison of gene content in closely related organisms
    • ID of specific organisms
21
Q

How is the Transcriptome measured?

A
  • qRT-PCR = Qualitative Real Time - PCR (only for individual genes)
  • Microarrays (for whole transcriptomes)
  • RNA-seq (for whole transcriptomes)
22
Q

Microarrays

A
  • small, solid supports to which genes or segments of genes are fixed and arrayed spatially in known pattern
  • gives a relative estimation of RNA abundance btwn 2 conditions
23
Q

Steps of using Microarrays:

A
  1. isolate RNA
  2. cDNA generation w/ PCR
  3. labeling of the diff samples
  4. hybridization of microarray (indicates transcribed gene)
  5. imaging
    1. when A>B
    2. when B>A
    3. when A=B
24
Q

RNA-seq

A
  • used to compare 2 conditions
  • collect all RNA from sample
  • convert to cDNA
  • sequence w/ nex-gen
  • align sequences to genome
  • count reads for each gene
  • compare samples
25
Q

Proteomics

A
  • study of all the proteins that are present in a cell under a given condition
    • what DOES get made
    • more informative
    • measure by: 2D-gels, Mass spec
26
Q

2D-Gels

A
  • techniqe for the separation, idenfication, and measurement of all proteins present in sample
  • Steps:
    • collect proteins
    • digest w/ trypsin (protease)
    • ionize them
    • pump through mass spec
    • match peptide fragments w/ ORF’s predicted from genome
    • compare samples for relative abundance
27
Q

Interactome

A
  • interaction maps that look at how all genes/proteins in cells react together
28
Q

Metabolomics

A
  • the complete set of metabolic intermediates and other small molecules produced in an organism
  • techniques: mass spec
29
Q

Paralogs

A

genes w/in an organism whose similarity to one or more genes IN THE SAME ORGANISM is the result of gene duplication

30
Q

Orthologs

A

genes found in one organism that are similar to those IN ANOTHER ORGANISM but differ b/c of speciation

31
Q

Xenologs

A

gene duplications that arise due to the result of horizontal gene transfer

32
Q

Core Genome

A

genes shared by all strains of the species

33
Q

Pan Genome

A

includes all the optional extras present in some but not all strains of the species

BIO 357 - Microbiology (8 decks)

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