midterm 2 Flashcards
microbiota vs microbiome
biota: defined environment
biome: microbes + genomes + environment
amplicon analysis function and general steps
-amplify regions that diverge among taxa
amplicons -> sequences -> bins -> taxonomy -> determine abundance
ISME
international society for microbial ecology
sample unit factors and considerations
-size, geographic unit, temperature, season
-rarefaction (new species linear/constant)
-uniformity (clusters evenly spaced)
plot of # samples vs species richness
-plateau = sampling completed, all diversity shown within that number of samples
types of sequencing
-sanger
-illumina (sequence by synthesis)
-amplicon
-shotgun metagenomics
-long-read (oxford nanopore)
-FISH
sanger sequencing steps
- mix ddNTPs w/ fluorescence, chain halts polymerase when incorporated
- gel with detector, measure fluorescence
- beginning/end unreliable (beginning= all hitting detector at once, end = ddNTP depleted)
illumina short read sequencing main steps
- library prep (amplicon or shotgun metagenomics)
- cluster generation
- sequencing by synthesis
amplicon sequencing steps
- isolate DNA
- PCR on sequence
- illumina ngs
- DADA2 analysis and ASV counts/taxa table
shotgun metagenomics sequencing steps:
- fragment isolates, select <500bp
- ligate to adaptor, denature strand and flow over flow cell
- amplify sequence bound to flow cell, one copy bound in place
- wash away one piece, other piece forms bridge forming clusters
- flow ddNTP with fluorescent, reverse by adding -OH
- image fluorescence and repeat
long read sequencing steps
- enzyme unwinds DNA, ssDNA fed into nanopore
- measure resistance across membrane bilayer
- 6nt in pore at a time
phenol-chloroform DNA extraction
-lysis buffer + phenol/chloroform/isomyl alcohol
-TRIS/EDTA/NaCl/SDS
-silica beads to homogenize sample
solid-phase DNA extraction
- lysis buffer -> silica column
- DNA wash buffer
- DNA elution buffer
illumina sequencing output
-fastq file
-ID, sequence, +, quality score (ASCII)
phred quality of illumina sequencing formula
phred quality = -10 log (p error)
=ord (Q char) - 33
OTU / operational taxonomic unit
-clusters that differ by a fixed threshold
-UPARSE, uclust
->97% similar = OTU
ASV / amplicon sequence variant
-learn error rates and correct mathematically
-allows variants of 1-2nt
-DADA2
what is DADA2 used for?
-read counts represent environmental abundance from illumina ngs output
DADA2 main steps
- illumina sequencing (amplicon or shotgun)
- analyze quality score, truncate and filter low quality
- learn error rates mathematically
- group replicate sequences, merge overlap forward/reverse reads
- blast and identify unique sequences, analyze taxonomy
what is chimera?
-artifact sequence
-2+ biological sequences incorrectly joined together
how can you analyze DADA2 abundance data?
directly:
-bar chart, pie charts, dimension reduction
statistically:
-alpha/beta diversity
shotgun metagenomics pipeline
community sample -> DNA extract -> fragment DNA -> illumina NGS -> Fcn profile, align kmers to reference kraken, or build contigs
whole genome sequencing pipeline
single colony -> DNA extract -> fragment DNA -> illumina NGS -> contigs
contigs vs mags
contigs: all reads from same organism
mags: all reads from different organisms
transcriptomics pipeline
single colony -> DNA extract -> delete rRNA -> convert to cDNA -> illumina NGS
metatranscriptomics pipeline
community sample -> RNA extract -> deplete rRNA -> cDNA -> illumina NGS
relative abundance plotting steps
- DADA2->ASV->taxonomy
- plot #reads/samples/experiments
- group by phyla or healthy vs diseased
- use PCA to find max variance
alpha diversity
-diversity within one sample
-richness, evenness, phylogenetic relatedness
simpson vs shannon alpha diversity
simpson: 0-1
shannon: 0-10
-weighting toward dominant vs minor species
beta diversity
diversity between microbial communities
bray-curtis beta diversity vs unifrac
bray curtis: quantitative measure / presence-absence of species
unifrac: bray curtis considering phylogenetic relatedness of species
methods of measuring community density
- flow cytometry
- 16S rRNA standard curve
- DNA quantification
carrying capacity
maximum density of organisms supported by an ecosystem, disctated by resource availability
steps for PCA
- center data around zero
- line through origin - PC1 explaining max variance
- keep finding PC’s unitl variance is 100%
kraken/braken functional analysis steps
- align reads to taxa
- fragment reads to kmers, associate with lowest common ancestor
- braken calculates taxa relative abundance
functional analysis pathways
-HUMAnN3
-bowtie2
-MetaPhiAn2
HUMAnN3 function
determine presence or absence of microbial pathways from metagenomic data
bowtie2 function
aligns kmers to functionally annotated pangemone of different species
functional analysis pathways overall goa/output
- gene family abundance
- pathway abundance
- pathway coverage
HUMAnN3 steps
- align reads to clade-specific marker
- align reads to pangenome
- blast extra proteins to determine function
niche definition
habitat, resources, and relationship between species allowing growth and reproduction
fundamental niche vs actual/realized
fundamental: range of possible growth conditions
actual: environment, nutrient availability, competitors, predators, and phage
niche differentiation vs construction
differentiation: overlapping niches coexisting
construction: alter niche and change survival chance
natural theory of community assembly factors
-dispersal (high = high alpha, low beta diversity)
-ecological drift
-pH, temperature, nutrients, host interactions, competition, etc
nutrient niche theory
ecological niche in gut defined by available nutrients and can only colonize if it can efficiently use limiting resource
colonization resistance
commensal microbes provide protection against pathogen colonization and infection
colonization resistance extended competition assay steps
- select 10 best isolates
- challenge community with pathogen, pass to new media, measure pathogen abundance
- analyzer higher-order effects
higher-order effects
studying effect of one species changing due to the presence of a 3rd party species
r-strategists
adapted to unstable environment, high reproductive rate
k-strategists
adapted to stable environment, low reproductive rate
r and k strategists coexisting results in:
-higher alpha diversity
-transit time altering diversity
biological clock and examples in mammals
physiological processes and geophysical time
-CLOCK & BMAL1 (light phase)
-cryptochromes and PER1/2 (dark phase)
PER1/2 knockout strain allows study of:
circadian rhythms
small intestine components
-high in sugars
-lower transit time
-select r strategists
large intestine components
-complex polysaccharides
-high transit time and density
-select k strategists
mucin glycoprotein functions
-use sugars released by glycosidases
-sulfated glycoproteins allow sulfate reducers
-sialyation allows sialidase release
immature biofilm
-degrade mucus and metabolize glycans/peptide backbone of mucin glycoprotein
-allows cross-feeding of dependent species
restaurant hypothesis
-e. coli persists if they were the first colonizers in gf mice, but if introduced to developed community they will not become established
-involves adhesion sites and priority effects
patchiness in gut caused by:
-association with mucosal community and luminal communities
dietary fibre gut microbiome purposes
- nutrient niches
- spatial structure
keystone species
large impact on the rest of the community, obligate or facultative dependence
competition
one individual decreases survival/reproduction of another
exploitative vs interference competition
exp: indirect, typically due to nutrients
int: direct harm
interference competition examples
-Type IV/IVSS
-antibiotics
-bacteriocidins, colicins, microcins, lantibiotics
mutualism/cooperation
bidirectional positive interactions and exhange of metabolic products
bacteriodota
-degrade complex polysaccharides
-extracellular degradation with outer glycoside hydrolases
stability / community complex relationship & competition
-higher complex community will be less stable
-higher competition is more stable
cooperation and adding competitive species relationship
-higher cooperation leads to lowered stability
-adding competitive species highers stability to a point before it depletes
