Sample Processing Genomics

1. Genetic markers used to track specific microbial populations or biological functions in the environment

Reporter genes (Bioreporters).

2. Four common examples of reporter genes mentioned in the notes

lacZ gene, xylE gene, lux gene, and Green Fluorescent Protein (GFP).

3. Analysis technique that focuses strictly on polar fatty acids

Phospholipid-Linked Fatty Acid (PLFA) analysis

4. Threshold for genomic DNA-DNA hybridization to consider two isolates as the same species

≥ 70% hybridization.

5. The most common gene used for amplification and sequence analysis in prokaryotes

16S rRNA gene.

6. The approximate size (in base pairs) of the DNA amplicon produced by the 27F/1492R primer pair

1,400 to 1,500 bp.

7. Major advantage of the culture-independent approach

It allows detection of microbial populations and activities without the need to grow them in the lab.

8. The two primary steps involved in nucleic acid recovery

Isolation of microbial cells followed by lysis, OR direct lysis in the environmental matrix.

9. Technique used for the in vitro replication of defined nucleic acid sequences

Polymerase Chain Reaction (PCR).

10. Modern technology used to identify populations and determine diversity by sequencing millions of fragments simultaneously

Next-Generation Sequencing (NGS).

11. The core process used to determine phylogenetic relationships in culture-independent studies

Amplification of molecular signature sequences followed by sequencing or hybridization.

12. Process involving sequencing DNA and comparing actual nucleotide sequences for comparable genes

Genetic Fingerprinting.

13. Method used to separate DNA fragments of the same length but different sequences

Denaturing Gradient Gel Electrophoresis.

14. How are sequences determined after a DGGE run?

By excision of the bands, PCR re-amplification, and sequencing.

15. Method that uses restriction endonucleases to cut DNA at specific recognition sequences

Restriction Fragment Length Polymorphism.

16. How are DNA fragments separated in RFLP?

Based on size by gel electrophoresis.

17. A more advanced version of RFLP used for community profiling

Terminal Restriction Fragment Length Polymorphism (TRFLP).

18. Method providing detailed microbial community analysis by targeting the internal transcribed spacer (ITS) region

Automated Ribosomal Intergenic Spacer Analysis (ARISA).

19. Location of the ITS region in the rRNA operon

Between the 16S rRNA gene and the 23S rRNA gene.

20. Why is the ITS region used in ARISA?

It is a region of variable length, meaning different species produce fragments of different sizes.

21. How are ARISA results visualized?

As "peaks" on a chromatogram, where each peak corresponds to a different community member.

22. Approach where all community DNA is sequenced to reconstruct genomes from the environment

Environmental Genomics (Metagenomics).

23. Limitation of the environmental genomics approach

Assembled genomes may not all be complete.

24. You want to identify a specific "unculturable" bacterium in a soil sample. Should you use a culture-dependent or culture-independent approach?

Culture-independent approach (e.g., 16S rRNA sequencing).

25. Critical Thinking: Why is the 16S rRNA gene called a "molecular chronometer"?

Because it is highly conserved across all prokaryotes and changes slowly over time, allowing scientists to track evolutionary relationships.

26. Application: If two bacteria show 65% DNA-DNA hybridization, are they the same species?

No, they must meet the ≥ 70% threshold.

27. Confusion: What is the difference between PLFA and FAME analysis?

FAME analyzes both polar and non-polar fatty acids, while PLFA focuses only on polar fatty acids (phospholipids).
Critical Thinking: Why does DGGE separate DNA if the fragments are the same size?

28. Application: A researcher uses a fluorescently labeled primer to amplify the ITS region. What specific method are they performing?

ARISA (Automated Ribosomal Intergenic Spacer Analysis).

29. Final Summary: Which question does NGS best answer: "Who is there," "How many," or "What are they doing?"

It primarily answers "Who is there" and "What is the diversity," though functional metagenomics can also answer "What are they doing."

30. The study of the total genetic material (the "metagenome") recovered directly from environmental samples

Environmental Genomics (Metagenomics).

31. The approach where all community DNA is sequenced, though the resulting genomes may be incomplete

Environmental sampling approach.

32. Three major bacterial repetitive sequences used in genetic fingerprinting

ERIC, REP, and BOX elements.

33. What does the acronym "ERIC" stand for?

Enterobacterial Repetitive Intergenic Consensus.

34. What does the acronym "REP" stand for?

Repetitive Extragenic Palindromic sequence.

35. Technique used to amplify sequences between repetitive elements to create a genomic "fingerprint"

rep-PCR.

36.

37. Approach that targets a specific gene, such as the 16S rRNA gene or a gene for ammonia oxidation

Single-gene community sampling approach.

38. Approach that involves restriction digestion of total DNA followed by shotgun sequencing or direct high-throughput sequencing

Environmental sampling approach.

39. Outcome: A phylogenetic snapshot of most members of the community

Single-gene approach outcome.

40. Outcome: Discovery of entirely new genes and identification of all gene categories in a community

Environmental (Metagenomic) approach outcome.

41. Outcome: Determining the phylogenetic diversity of a specific microbial guild (e.g., ammonia-oxidizing archaea)

Single-gene approach outcome.

42. Outcome: Linking specific functional genes to specific phylotypes (who is doing what)

Environmental (Metagenomic) approach outcome.

43. Advantage of sequencing directly (without cloning) using high-throughput sequencers

It reduces bias and allows for a more comprehensive "gene pool" analysis.

44. Application: You want to find a brand-new enzyme that can degrade plastic in the ocean. Which approach should you use?

Environmental (Metagenomic) approach (to discover new genes).

45. Critical Thinking: Why might a "Single-gene" approach give a "snapshot" but not a "total picture" of an ecosystem?

Because it only looks at one conserved gene (like 16S) to identify who is there, but doesn't tell you the full metabolic potential of those organisms.

46. True or False: In the environmental approach, every genome assembled is guaranteed to be 100% complete.

FALSE; assembled genomes from the environment are often incomplete.

47. Application: A researcher uses the BOX element to differentiate two strains of E. coli. What method are they using?

rep-PCR (using repetitive sequences).

48. Confusion: What is the difference between a "Phylotype" and a "Guild"?

A phylotype refers to organisms grouped by genetic similarity; a guild refers to organisms that perform the same functional role (e.g., ammonia oxidizers) regardless of their phylogeny.

49. Critical Thinking: Why is the ITS region in ARISA considered more "detailed" for community analysis than the 16S gene alone?

The ITS region is more variable in length and sequence between closely related species, allowing for better differentiation at the species or strain level.

50. Application: You are looking for "Ammonia Oxidizing Archaea" specifically. What gene should you amplify?

A functional gene encoding a specific function, such as the amoA gene (ammonia monooxygenase).

51. True or False: Metagenomics allows you to link a specific metabolic function to a specific organism without ever growing that organism in a lab.

TRUE; this is the core benefit of the "linking genes to phylotypes" outcome.

52. Scenario: You have a community DNA sample and you perform "Shotgun Sequencing." What is your primary goal?

To capture the "Total gene pool" of the community.

53. Final Oral Prep: Explain the difference in the "Outcome" of a 16S rRNA study vs. a Metagenomic study.

16S rRNA studies tell you "Who is there" (Phylogenetic snapshot); Metagenomic studies tell you "What they can do" (Total gene pool/Discovery of new genes).

54. What is the outcome of single-gene phylogenetic tree

(1) phylogenetic snapshot of most members of the community (2) phylogenetic diversity of a microbial guild such as the ammonia-oxidizing bacteria archaea

55. What is the outcome of the total gene pool of the community

(1) identification of all gene categories (2) discovery of new genes (3) linking of genes to phylotypes