Chapter 18: Microbial Genomics

Question 1

DNA Sequencing Methods: Sanger DNA Sequencing

(1) DNA sequencing
Components:
* Template DNA
* Oligonucleotide primer
* DNA polymerase
* Deoxynucleoside triphosphates (dNTPs)
* (2)

In such a reaction mixture, DNA synthesis continues until a (2) , rather than a dNTP, is added to the growing chain.
* Without a (3) to attack the (4) of the next dNTP to be incorporated, synthesis stops.

Answer 1

1. Chain-termination
2. Dideoxynucleoside triphosphates (ddNTPs)
3. 3’-OH group
4. 5’-phosphate group

Question 2

Sanger Method

DNA synthesis continues until a ddNTP, rather than a dNTP, is added to the growing chain.
* Without a 3′-OH group, synthesis stops.

(1) reactions prepared, one for (2).
* Results in a collection of DNA fragments of varying lengths, each ending in the same ddNTP.

Samples prepared for automated sequencing.

Answer 2

1. Four separate synthesis
2. each ddNTP

Question 2

Automated Sanger DNA Sequencing

• Uses four different colored (1).
• (2) determines order.
• Results are recorded on a (3).

Answer 2

1. fluorescent labeled ddNTP
2. Electrophoresis and laser beam
3. chromatogram

Question 3

Next-Generation DNA Sequencing (NGS)

(1) sequencing techniques—thousands of identical DNA fragments are sequenced simultaneously.

Uses (2) templates attached to a solid substrate.

Makes genomic sequencing faster and cheaper.
* Avoids need to insert individual DNA fragments into vectors.

Answer 3

1. Massively parallel
2. short, sheared pieces of DNA

Question 4

Sequencing By Synthesis

Identifies each nucleotide as (1).
Synthesis uses a modified fluorescent nucleotide that stops the reaction because the (2).
* Nucleotide does not (3) until it is incorporated into growing DNA strand.

Answer 4

1. it is incorporated
2. 3′-OH is blocked
3. fluoresce

Question 5

Whole-Genome Shotgun Sequencing

Four stage process:
* (1)—generates clones of portions of genome.
* (2)— determines sequences of genome fragments in vector.
* (3)—computer analysis joins overlap regions to form contig.
* (4)—proofreading ensures all reads of the same sequence are identical.

Answer 5

1. Library construction
2. Random sequencing
3. Fragment alignment and gap closure
4. Editing

Question 6

Next-Generation Genomic Sequencing

Depth of coverage and breadth of coverage improved.
* Coverage— (1) sequenced in a genome.
– 18-100 times is (2).
– (3)—high average number of reads.

Great depth of coverage increases accuracy.
* Decreases the chance that sequence data generated is from (4).

Answer 6

1. average number of times each nucleotide is
2. how often nucleotides are read
3. Deep sequencing
4. a random mistake in the polymerase activity

Question 7

Single-Cell Genomic Sequencing

Femtograms of DNA from a cell are (1).

(2)
* Occurs at single temperature and uses the DNA polymerase from bacteriophage phi29 to synthesize new DNA.

Answer 7

1. amplified to mg of DNA required for sequencing
2. Multiple displacement amplification (MDA)

Question 8

Metagenomics Provides Access to Uncultured Microbes

Metagenomics
* Study of microbial genomes based on (1).
* Used to learn more about the diversity and metabolic potential of microbial communities.
– Each DNA fragment comes from a collection of genomes found in a (2).
* Established the Genomic Encyclopedia of Bacteria and Archaea project.
– Improves reference databases by sequencing genomes of a wide variety of cultured microorganisms.

Answer 8

1. DNA extracted directly from the environment
2. microbial community, not a single organism

Question 9

Bioinformatics

(1)
* Bioinformatics combines biology, mathematics, computer science, and statistics to convert raw nucleotide data into (2).

Further examination carried out using (3).

Answer 9

1. Genome annotation
2. the location and potential of genes
3. in silico analysis

Question 10

Genome Annotation

Process that locates genes in the genome map.

Identifies (1).
* must be (2) that is not interrupted by a (3).
* must have (4) at the 5’ end and (5) at the 3’ end.

Answer 10

1. open reading frame
2. > 100 codons
3. stop codon
4. ribosomal binding site
5. terminator sequences

Question 11

Bioinformatics—ORFs and BLAST

ORFs that appear to encode protein are called (1).

(2)
* Base-by-base comparison of 2+ gene sequences.
* Assign tentative function of gene or protein structure.

Answer 11

1. coding sequences (CDS)
2. BLAST (basic local alignment search tool)

Question 12

Gene/ORF Terminology

(1)—genes from different organisms with similar ORFs.

(2)—two or more genes with very similar nucleotides sequences, likely from a duplication event.

(3)—naming standards for proteins and their motifs based on similarities among orthologous proteins.

Proteins that don’t align with known amino acid sequences fall into two categories.
* (4)—match known sequences in databases, but don’t have an assigned function (yet).
* (5)—products of genes unique to that organism.

Answer 12

1. Orthologues
2. Paralogues
3. Gene ontology
4. Conserved hypothetical proteins
5. Proteins of unknown function

Question 13

Functional Genomics Links Genes to Phenotypes

Functional Genomics

Puts genomic information in a (1).

Provides information on:
* (2)
* (3)
* (4)

Answer 13

1. biological context
2. metabolic pathways
3. transport systems
4. regulatory and signal transduction mechanisms

Question 14

DNA Microarray Analysis

Determines which genes are expressed at a specific time.

Arrays are (1) -> organized as a grid.

Each DNA spot (2) represents a single gene.
* (2) is usually a PCR product generated from a (3).

Answer 14

1. solid supports to which DNA is attached
2. probe
3. gene of interest

Question 15

Analysis of Gene Expression Using Microarrays

Based on hybridization between the (1) and the (2) from the microbe of interest.
* Reverse transcriptase converts mRNA to cDNA.
* cDNAs are (3) and incubated with the microarray.
* (4) is washed off.
* The microarray is scanned with (5) which indicates that hybridization has
occurred.

Answer 15

1. probe DNA
2. complementary sequences
3. labeled with fluorescent dyes
4. Unbound cDNA
5. laser optics to detect fluorescence

Question 16

RNA-Seq Method for Transcriptome Analysis

NGS method quantifying mRNA levels by measuring the “reads” matching each gene.

Cellular mRNA is converted to cDNA by (1).

cDNA are then (2).

Sequence data can be used in two ways:
* If sequenced genome exists, the cDNA are (3).
* If no reference genome, nucleotide sequence is (4).

Answer 16

1. reverse transcriptase
2. sequenced using NGS
3. aligned to reference genome
4. converted to amino acid sequences and compare to databases of known protein sequences

Question 17

Hierarchical Cluster Analysis

Organization of transcriptomic experiments.
Groups genes according to ().
E.g.
* Upregulated—red
* Downregulated—green
* Unchanged—black

Answer 17

level of expression

Question 18

Metatranscriptomics

Extraction of RNA (1), followed by sequencing and comparison to known sequences.
Describes transcriptome of (2).
* Yield transcripts that map to multiple reference genomes as well as some that fail to align with any reference genome.
* Novel transcripts represent newly discovered genes.

Answer 18

1. directly from the environment
2. an entire ecosystem

Question 19

Systems Biology: Making and Testing Complex Predictions

(1) with the molecular interactions that become pathways for catabolism, anabolism, regulation, behavior, environmental responses, etc.
* (2) of cells.
* May be important in studying (3).

Answer 19

1. Integration of “parts lists” of cells
2. Holistic study
3. host microbe interactions

Question 20

Comparative Genomics

(1) inferred by studying similar nucleotide and amino acid sequences among organisms.

Comparisons of genomes of strains within species and among species.
* (2)—genes that are no longer functional.

Comparative genomics is the tool by which virologists and evolutionary biologist have tracked the development of new strains of SARS-CoV-2.

Answer 20

1. Gene function and evolutionary relationships
2. Pseudogenes

Question 21

Comparative Genomics In Microbial Groups

• Horizontal gene transfer (HGT) found to be frequent.
• (1)—permanently integrated mobile genetic elements.
• Pathogenicity islands—virulence proteins.
• (2)—order of genes on genome.

Answer 21

1. Genomic islands
2. Synteny

Question 22

HGT Impacts Microbial Evolution

Core genome—set of genes found (1).
* Often thought of as the (2).
Pan-genome—(3) in a strain of a species.
* More recently acquired genes that enable (4).

Answer 22

1. in all members of a species
2. minimum genes needed to survive
3. every gene
4. microbial colonization of new niches