Lesson 1 of 13 · 11 min
What Metagenomics and the Microbiome Are
Metagenomics is the culture-independent sequencing of all the DNA extracted directly from a microbial community. Instead of growing one organism in the lab, you sequence the pooled genomes of everything present in a sample at once. This gives access to the large majority of environmental microbes that have never been grown in pure culture.
Single-isolate genomics starts from a pure culture: one strain grown on a plate, its DNA sequenced and assembled into a single genome. Metagenomics skips the culturing step and works on the mixed DNA of the whole community, so one dataset can contain hundreds of taxa at very different abundances.
Microbiome Vocabulary
A taxon is any named group of organisms, from a broad phylum down to a single species or strain. The microbiome is the full community of taxa living in a defined environment together with the genes they carry. Communities are usually called host-associated when they live on or in a host, such as the human gut or a plant root, and environmental when they live in habitats like soil, seawater, or a wastewater bioreactor.
Three Core Questions
Most metagenomic studies come down to three questions. Who is there identifies which taxa are present, how much measures their relative abundance, and what can they do asks which genes and metabolic pathways the community encodes. The first two describe composition, while the third describes functional potential.
Tip: Who is there and how much are composition questions that a single marker gene can answer well. What can they do is a function question that generally needs shotgun data, because a marker gene tells you an organism is present but not which metabolic genes it actually carries.
Amplicon Versus Shotgun
Amplicon sequencing targets one marker gene, most often the bacterial 16S rRNA gene, by PCR-amplifying a short variable region with conserved primers; it answers who and how much cheaply but says little about function. Shotgun metagenomics instead fragments and sequences all DNA in the sample, so it can profile taxa, gene content, and pathways together, at higher cost and heavier computation. The rest of this course returns to both strategies repeatedly, so it helps to know which question each one is built to answer.
# Count raw reads carrying the 16S rRNA 515F primer (Y = C or T, M = A or C)
zcat amplicon_R1.fastq.gz | grep -E -c 'GTG[CT]CAGC[AC]GCCGCGGTAA'47105Try it yourself: Grab one 16S amplicon FASTQ and one shotgun metagenome FASTQ from a public run, then run the grep -E -c command above on each. As long as the amplicon reads still carry their primers (they were not trimmed during demultiplexing), the amplicon file returns a hit count close to its total read count, while the shotgun file returns almost none, because shotgun reads begin at random genomic positions rather than at a fixed primer.