L4

why studying microbiomes is difficult

Microbiomes are always changing. They are dynamic

Most microbial species live as members of complex microbial communities

• Not as pure cultures
• Complex interactions among members, community composition is dynamic, depends on changing conditions

functional analysis

Not great either, because microbiomes acquire different functions, an enzyme of a seaweed breaking compound can be found in different micrbiomes

DNA sequence analysis

We can use DNA sequence information to identify species and tell us phylogenetic differences

• Each species might have a specific sequence of a ubiquitous gene

• E.g., SSU rRNA gene (16S rRNA in prokaryotes)

Amplicon Sequencing (16S/18S rRNA analysis)

• What it does: It amplifies fragments of the ribosomal small subunit (SSU) RNA gene and uses high-throughput sequencing.

• What it identifies: It creates a community profile to tell you which species are present and in what abundances.

• Does it identify members of the community? Yes.

Two techniques: Quantitative (microarrays) or semi-quantitative (massively parallel sequencing)

(Shotgun) Metagenomic Sequencing

• What it does: It takes the whole community DNA, fragments it, sequences it, and uses annotation algorithms to find functional genes.

• What it identifies: It looks at community functions to tell us what the community can do metabolically.

• Does it identify members of the community? While it sequences all DNA, its primary purpose is to identify what the community can do metabolically rather than just who is there. But it can identify who is there

Metatranscriptomics (RNA-Seq)

• What it does: It isolates and sequences the total pool of primarily mRNAs from the community, requiring a lot of reads because mRNA expression ranges wildly.

• What it identifies: It uses computer algorithms to annotate sequenced RNAs to reveal expressed microbial function (What gens are there)

• Does it identify members of the community? No, it focuses on identifying which genes are actively being expressed.

Metabolomics

• What it does: It analyzes the chemical environment, though it is limited in how many metabolites can be determined.

• What it identifies: It determines what microbes actually produce and consume, as well as possible cross-metabolic effects.

• Does it identify members of the community? No, it strictly looks at the metabolites (the products and consumed materials) in the community.

Why is 16S/18S SSU rRNA such a popular choice?

• Ubiquitous, exists in every organism

• Similar but unique sequence among all species

• Mosaic structure: variable and conserved regions

What are the two distinct tools used to interrogate the SSU rRNA gene?

• Primers: They are designed to amplify rRNAs from the majority of different species within a clade. This acts as the copying step for sequencing.

• Probes: They are designed to target regions unique to a phylogenetic clade (a branch on a phylogenetic tree). This acts as a direct detection tool to find a specific sequence.

What are the steps for analyzing a microbial community using DNA Sequencing?

• Step 1: Isolate DNA from the cells.

• Step 2 (The Rule): Use PCR to amplify the gene encoding SSU rRNA. Crucial Rule: The primers must be designed for the conserved regions. Because these regions are similar across all species, the primer acts as a universal key to amplify rRNAs from the majority of different species all at once.

• Step 3: Perform DNA sequencing on those amplified genes to read the unique variable regions locked inside.

• Step 4: Compare each acquired sequence against a database to find matches, generating a profile of which species are there and in what abundances.

If we interrogate enough rRNA genes from a microbial community, this will generate a community profile. Which species are here and in which abundances

How do Probe methods work, and what do they target?

• The Rule: Probes are designed to perfectly match variable regions (unique to a specific phylogenetic clade) so they only bind strongly to that specific target sequence.

• The Targets: Probes can target either 16S rRNA genes (to identify species) OR functional genes (to measure functional gene abundance, like on the GeoChip).

• Application 1 (Microarrays): Probes to hundreds of different genes are printed or built directly on the surface of a glass wafer.

• Application 2 (FISH): A fluorescently labeled probe hybridizes to a particular nucleic acid sequence directly inside fixed cells (no PCR copying needed!). This allows researchers to identify and visualize specific cells under a fluorescent microscope.

The Goal of Probes: using probes to identify specific clades or branches on a phylogenetic tree.

High-Throughput Sequencing (HTS)

• What it does: Produces millions of nucleic acid reads simultaneously in a single, highly parallel run.

• How it works: Relies on DNA polymerase chemical reactions during replication, detecting each new nucleotide incorporation by measuring pyrophosphate or hydrogen ion (H^+) release.

• Is it good or not: Excellent for community studies because it can sequence millions of pieces at once, though specific platforms vary in cost and error rates.

Sanger Sequencing

• What it does: Sequences DNA using older, traditional chain-termination chemistry.

• How it works: Processes individual reactions sequentially rather than in a massively parallel format.

• Is it good or not: Not good for community studies; it is not used because its throughput is far too low for complex microbiomes.

Illumina

• What it does: High-throughput sequencing of millions of nucleic acid fragments simultaneously.

• How it works: Detects the addition of each nucleotide using a specific fluorescent dye.

• Is it good or not: Very good for high-throughput data and widely used, but the chemical dyes used make it expensive

Ion Torrent

• What it does: High-throughput sequencing of nucleic acid fragments.

• How it works: Detects the release of hydrogen ions (H^+) as new chemical bonds form during strand synthesis.

• Is it good or not: Good alternative for high-throughput sequencing using semiconductor chip technology to measure electrical/pH changes instead of optical light.

PacBio & NanoPore (Long-Read Sequencers)

• What it does: Sequences nucleic acids with a focus on length rather than sheer short-read volume.

• How it works: Generates very long continuous reads of DNA molecules.

• Is it good or not: Good for resolving complex genomic structures, but has a lower total output, Very long reads. and a higher expense per analyzed nucleotide

What is the fundamental rule of how a microarray detects DNA?

• It is strictly a binding assay (no new DNA is synthesized/built during the test).

• It relies on nucleic acid double-strand formation (hybridization). This means you take single-stranded sample DNA that you copied beforehand, wash it over the chip, and it zips together with the single-stranded probe only if they are a perfect match.

What are the two ways Microarrays are built, and what are their pros and cons?

• Spotted Arrays: Probes are printed or dropped onto a glass wafer as liquid. Pros: Flexible to design. Cons: High variability in spot size and probe density.

• Fabricated Arrays: Probes are chemically built directly onto the surface using photolithography. Pros: Very high probe density and very low variability (every chip is identical).

What does a Phylogenetic Microarray target, and what question does it answer?

• Target: 16S rRNA genes.

• Analysis: It tells you who is in the community by identifying specific clades or species (often resulting in a heatmap of relative abundance for groups like Firmicutes or Bacteroidetes).

What does a Functional Microarray target, and what is the GeoChip?

• Target: Functional genes (or mRNA expression), rather than rRNA.

• Question: It tells you what the community can do by measuring functional gene abundance.

• GeoChip Example: A massive functional array (like GeoChip v5) that contains 167,000 probes targeting specific functional genes, such as those responsible for nitrogen cycling or metal resistance in soil.

What is Quantitative PCR (qPCR) and what is its main advantage?

• Function: It amplifies DNA while measuring it in real-time using fluorescent dyes or probes.

• The Advantage: It is highly targeted and quantitative. It calculates exactly how many copies of a specific target DNA sequence you started with. (Note: To determine the actual number of bacterial cells, you must divide the total DNA copies by the gene's specific "copy number" per genome).

How can qPCR be used to find one specific species versus a mix of species?

• Single Target: You use primers/probes designed for a variable region unique to one species. If the graph goes up, that exact microbiome is there.

• Multiplexing (The Mix): You put primers/probes for multiple different species in the same tube, each attached to a different colored fluorescent dye (e.g., green for E. coli, red for Salmonella). The machine tracks the different colors simultaneously.

What happens if you use conserved primers in qPCR instead of specific ones?

• Universally Conserved (e.g., 16S rRNA): Amplifies all bacteria. This tells you the Total Bacterial Load of the entire sample, regardless of species.

• Clade-Specific Conserved: Amplifies only a specific evolutionary family (e.g., a photosynthesis gene unique to Cyanobacteria). It acts as a universal primer, but strictly for that one group.

What are the three phases of a qPCR fluorescence graph?

• Lag Phase: The flat beginning; the reaction just started and there isn't enough DNA to detect yet.

• Exponential Phase: The steep upward curve; DNA is perfectly doubling every cycle. This phase is what the machine uses to calculate your exact starting amount.

• Plateau Phase: The flat top; the reaction runs out of ingredients (like nucleotides or primers) and stops growing.

What does in situ mean, and how does it make FISH different from qPCR or sequencing?

• Meaning: In situ means "in its original place."

• The Difference: Instead of grinding up the sample to extract the DNA, you keep the cells completely intact. You send the fluorescent probes inside the whole cells to find their targets.

What are the main steps to perform FISH on a sample?

• 1. Fixation & Permeabilization: Chemically lock the cells in place and poke microscopic holes in the cell membrane.

• 2. Hybridization: Flood the sample with fluorescent probes that swim through the holes and bind to complementary targets.

• 3. Wash & Look: Wash away unbound probes and view the intact, glowing cells directly under a fluorescence or confocal microscope.

Why does FISH usually target rRNA instead of a specific DNA gene?

The Brightness Factor: A bacterium might only have one copy of a specific DNA gene (which is too dim to see). But an active cell has thousands of ribosomes made of rRNA. Targeting rRNA allows thousands of probes to bind inside a single cell, making it glow incredibly bright!

What is the unique advantage of FISH?

• Spatial Architecture: Because you don't destroy the sample to extract the DNA, FISH tells you exactly WHERE the microbes are. It allows you to see the physical structure of communities (like biofilms) and which species live next to each other.

How can FISH be used to distinguish between different species in the exact same sample?

• The Method: You use multiple probes at the same time, each tagged with a different colored fluorescent dye.

• The Result: You might use a red dye for a probe targeting E. coli and a green dye for a probe targeting all Bacteria. Under the microscope, you can visually separate the specific species (red) from the rest of the general community (green) all in one single image.

If FISH lets us "look" at the cells, do we still need a computer?

• Advanced Imaging (Confocal): When using confocal microscopy, human eyes can't build 3D models. The computer software takes the optical slices gathered by the microscope and renders the full 3D spatial architecture of the community.

• Quantitative Analysis: The computer is required to calculate the actual biovolume. It digitally analyzes the image to measure exactly how much area the glowing pixels take up, turning visual fluorescence into hard, quantifiable data.

The computer can identify which genus each cell
belongs to. And can also calculate distances between cells.