MICR 4040: How Do We Study Microbes?

What is microbial ecology studying?

The interactions, diversity, distribution, and functions of microorganisms in their environments.

What revolutionized microbial ecology?

High-throughput sequencing (HTS), which makes it possible to study whole microbial communities using molecular and meta-omic methods.

Why did traditional cultivation reveal limited microbial diversity?

Most environmental microbes do not grow under standard laboratory conditions; cultivation historically accessed less than 1% of microbial diversity.

What is cultivation useful for?

Isolating living microbes for mechanistic studies, genome sequencing, functional assays, and experimental testing.

What is culturomics?

High-throughput cultivation: samples are diluted into many wells, grown under varied conditions, and colonies are sequenced.

What are the main strengths of culturomics?

It provides living isolates and experimental material, expands reference databases, and supports genome and functional studies.

What are the main limitations of culturomics?

It is costly and labor-intensive and remains strongly biased by growth media and conditions.

What is amplicon sequencing?

Sequencing PCR-amplified marker genes from environmental DNA to profile microbial community composition.

Which markers are commonly used in amplicon sequencing?

16S rRNA for bacteria and archaea; 18S rRNA or ITS for fungi.

What is the basic amplicon-sequencing workflow?

DNA extraction → PCR with primers → sequencing → OTU clustering or ASV denoising.

What are OTUs?

Operational taxonomic units: sequence clusters grouped by a similarity threshold to represent microbial taxa.

What are ASVs?

Amplicon sequence variants: exact, error-corrected sequence variants inferred by denoising.

How do ASVs differ from OTUs?

ASVs provide finer and more reproducible sequence resolution; OTUs group similar sequences using an arbitrary similarity cutoff.

What are common amplicon-analysis tools?

QIIME2, USEARCH/VSEARCH, and DADA2.

What are the advantages of amplicon sequencing?

It is inexpensive, scalable, fast, requires little biomass, and is useful for broad community surveys.

What are the limitations of amplicon sequencing?

PCR and primer bias, often genus-level resolution, and indirect rather than directly measured functional information.

What is functional inference from amplicon data?

Predicting possible functions from taxonomic marker data using tools such as PICRUSt, Tax4Fun, or FAPROTAX.

What is metabarcoding?

Using DNA barcode sequences to identify many organisms or taxa in a mixed environmental sample.

When is amplicon sequencing or metabarcoding appropriate?

When the main question is who is present or how community composition changes, especially with limited budget or biomass.

What is shotgun metagenomics?

Sequencing all DNA in a sample without targeting one marker gene.

What can shotgun metagenomics reveal?

Higher-resolution taxonomy, genes and pathways, viruses, rare taxa, and metagenome-assembled genomes (MAGs).

What is a MAG?

A metagenome-assembled genome reconstructed from DNA sequences obtained directly from a mixed community.

What is the shotgun metagenomics workflow?

DNA extraction → quality control and host-read removal → assembly → binning → annotation.

What tools may be used in shotgun metagenomics?

Trimmomatic/Bowtie2 for quality control and host removal; MEGAHIT/metaSPAdes for assembly; MetaBAT/MaxBin for binning; HUMAnN2/MEGAN/Prokka for annotation.

What are the strengths of shotgun metagenomics?

It captures taxonomy and function, is culture-independent, and can detect viruses and rare organisms.

What are the limitations of shotgun metagenomics?

It is expensive, data-heavy, computationally demanding, and sensitive to contamination and pipeline choices.

What is metagenomics best for?

Determining who is present and what genes or functional capabilities the community contains.

What is genome assembly?

Reconstructing longer genomic sequences, called contigs or scaffolds, from shorter sequencing reads.

What is de novo assembly?

Building genome sequences from reads without relying on a reference genome.

What is resequencing or reference-guided assembly?

Aligning reads to a known reference genome to identify variants and genome differences.

What is metatranscriptomics?

Sequencing expressed RNA, especially mRNA, to measure active gene expression in a community.

What does metatranscriptomics add beyond metagenomics?

It indicates which genes are being expressed, not merely which genes are present.

What are challenges of metatranscriptomics?

Host RNA contamination, RNA instability and handling demands, and high cost.

What is metaproteomics?

Using mass spectrometry to identify and quantify proteins in a microbial community.

What does metaproteomics reveal?

Which proteins and enzymes are actually present and their relative abundance.

What is metabolomics?

Profiling small-molecule metabolites produced or modified by organisms and their environment.

What does metabolomics reveal?

The chemical products of biological activity and links between microbial activity and ecosystem processes.

What is peptide mass fingerprinting?

Identifying proteins by comparing mass-spectrometry peptide patterns with reference databases.

What is multi-omics integration?

Combining genomic, transcriptomic, proteomic, and metabolomic data for a more complete view of a biological system.

What does the evidence ladder ask about a microbiome?

Who is there; what they can do; what they are doing; and whether the activity is actually occurring.

Which method answers “Who is there?”

Marker-gene profiling such as amplicon sequencing or metabarcoding, and also shotgun metagenomics.

Which method answers “What can they do?”

Shotgun metagenomics, because it profiles genes and pathways.

Which method answers “What are they doing?”

Metatranscriptomics, which measures expressed genes.

Which method answers “Are they really doing it?”

Metaproteomics and metabolomics provide evidence of proteins, enzymes, and chemical outputs.

What factors are critical in experimental design?

Adequate sample size, biological replication, metadata, controls, technical replicates, and consistent sample storage and extraction.

Why is metadata important?

It records variables such as diet, host genotype, soil chemistry, and other context needed to interpret microbial patterns.

Why are blanks and technical replicates important?

Blanks help detect contamination; technical replicates assess measurement and processing variability.

Why must extraction methods be consistent?

Different DNA or RNA extraction kits can introduce systematic biases and change observed community profiles.

What supports reproducibility in microbial ecology?

Standardized workflows, open-access tools, benchmarking, complete metadata, and transparent data submission.

What is CAMI?

Critical Assessment of Metagenome Interpretation, a benchmarking effort for evaluating metagenomic analysis pipelines.

How does method choice depend on the research question?

Choose based on whether you need taxonomy, genetic potential, active expression, proteins, metabolites, or an integrated view, while considering budget and sample type.

What did the Crohn’s disease case example show?

Amplicons identified broad taxonomic shifts; metagenomics found carbohydrate-metabolism differences; metatranscriptomics showed active inflammatory-pathway expression.

What is the central takeaway from the lecture?

Cultivation remains valuable but limited; amplicons are cheap and fast but lower resolution; metagenomics is detailed but costly; and meta-omics add functional layers.