12) Techniques in Microbiome Studies Flashcards
define microbiome
microorganisms in a particular environment or combined genetic material of the microorganisms in a particular enviornment
define microbiota
ecologial community of commensal, symbiotic and pathogenic MOs found in and on all multicellular organisms studies to date from plants to animals (not walls, desks and rocks)
what are 3 techniques used in microbiome semantics?
- 16S rRN targeted Amplicon Sequencing
- Metagenomics
- Single-cell Genomics/Read Cloud sequencing
describe 16S rRNA targeted amplicon sequencing
what does this technique tell us?
- amplifying and sequencing 16S rRNA genes from a sample
- tells us which bacteria are inhabiting in an environment
describe metagonimcs techniques
what does this technique tell us?
- sequencing all genetic material from an environment
- tells us the functional potential of a microbial community with little or not info on individual organisms
describe single-cell genomics/read cloud sequencing
what does this technique tell us?
- sequences the genetic material from a microbial community
- can be done one cell at a time or after labelling individual chromosomes
- provides detailed genomic info about the most abundant individual organisms in a community
how many chormosomes do bacteria have?
most have a single chormosome not in a nucleus
but some bacteria can have two
describe the size of chromosomes in bacteria
can vary
but 5-million base pairs is the average
can bacteria have extra-chromosomal DNA?
example of this?
yes, like plasmids
describe mobile genetic elements
when bacteria are capable of transferring genes to other bacteria
how many 16S rRNA genes does each bacteria have?
has at least one
some bacteria have several
what is 16S rRNA gene? what is it’s role?
a gene present in all prokaryotes, but not present in any eukaryotes
it is like a barcode telling you what the identity of the bacteria is
what is the 16s rRNA gene used for? why?
- phylogenic studies for bacterial identification
- suggested that the gene is a reliable “molecular clock” b/c it has hyper variable regions useful for phylogenetic identification of bacteria
what is 16S rRNA targeted Amplicon Sequencing used for?
to sequence entire bacterial populations only in terms of composition
why is 16s rRNA sequenced in microbiome studies? (2)
- it has slow evolution, thus sequence can be linked to taxonomy accurately (in other words, sequencing wouldn’t work with genes that evolve often)
- it is currently the only used taxonomic marker compiled in databases with reliable taxonomic information
what are limitations of 16s rRNA targeted Amplicon Sequencing?
- taxon recovery tests using 16S rRNA gene sequences show that short reads are not suitable for accurate richness estimations and accurate classifications of high taxa
- not all bacterial and arcahel species have been classified yet. It would take over 1000 years to classify remaining species
- bacterial genomes are 5 million base pairs, but 16S rRNA genes are sequenced only 250 base pairs
when should 16S rRNA sequencing be used? (2)
- to compare bacterial populations between two or more different “things” (ie. diseased state and healthy state)
- to determine how the bacterial population changes over time or in response to an event
when should another technique be used instead of 16S rRNA sequencing?
what should be used instead?
- to observe changes in abundance for a gene or species. Use culture genomics, rt-pcr.
- to elucidate functional potential of the population. Use metagenomics/ transcriptomics
- to determine functional potential of individual organisms in the population. Use culture sequence, single cell genomics, read cloud sequencing
describe the theory behind metagenomics
cutting all DNA from an entire community into small pieces to sequence it, then putting in back together
what does metagenomics tell you?
what does it not tel you?
functional potential of a microbial community
does not tell you which bacteria does what
what is PICRUSt?
what does it depend on?
- provides estimate of genes present in the metagenome based on 16S rRNA targeted amplicon sequencing
- depends on availability of sequenced genomes from well-studied environments
compare 16S rRNA targeted amplicon sequencing vs metageonomic sequencing
16S rRNA
- cheaper per sample
- can be sequenced by MiSeq, HiSeq, MinION
- uses variety of processing softare: RDP pipeline, QIIME, mothur
- uses variety of interpretation software: R, microbiome analyst
metagenomic seq:
- more expensive per sample
- HiSeq
- variety of ways to analyze data (denovo assembly, mapping to genomes and functional genes databases)
what are problems that may occur in 16S rRNA sequencing due to poor sequencing strategy?
- if reads don’t fully overlap, then errors in the 5’ read will not be corrected by the 3’ read
- if you assume that each error generates a unique read, this results in new OTUs (operational taxonimic unit used to categorize bacteria based on sequence similarity) just based on error
what is a confounder?
variable or factor that influences the dependent and independent variable
correlation does not equal _____
what is an example of this?
causation
example:
- there is a difference b/w intestinal microbiome of children with autism and those without
- children with autism statistically have higher sugar, gluten and dairy free diets. But studies rarely control for differences in diet
- is the change in the microbiome linked ot autism? or is the changes in diet a result of the autism?
what are errors that may occur due to DNA extraction?
each method/kit will extract a slightly different population
some portions of the population will be missed
meta-studies often ignores this and assumes all DNA is extracted the same way
what is Mothur?
what does it not do?
what does it mean when a study uses Mother?
- data processing method
- does not sequence reads that don’t fully overlap (which may occur due to poor sequencing)
- when Mothur is used, this means that sequencing was done well
what is QIIME?
- data processing method
- does not require that reads fully overlap
- thus, poorly sequencing with many errors may still produce a result
