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Food Microbiology > Techniques in Microbiome Study > Flashcards

Techniques in Microbiome Study Flashcards

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1
Q

True/False: sequencing a microbiome is very costly

A

False; can be done today for $18

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2
Q

What is the difference between a microbiome and microbiota?

A

microbiome: collection of microbes living on a larger organism/part of body/particular environment (or their combined genetic material)
microbiota: combination of all known microbiomes from living creatures

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3
Q

the 3 techniques important for microbiome study:

A

16S Targeted Amplicon Sequencing
Metagenomics
Single cell genomics/Read Cloud sequencing

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4
Q

Describe the general function of 16S Targeted Amplicon Sequencing, and what it is good for:

A

16S RNA genes from microbiome are AMPLIFIED and SEQUENCED

good for identifying which microbes are present

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5
Q

Describe the general function of Metagenomics, and what it is good for:

A

sequence ALL GENES present -> good for identifying FUNCTIONAL POTENTIAL (what metabolism occurs, etc) but does not give info on what microbes are present (what genes belong to which microbes)

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6
Q

Describe the general function of Read Cloud Sequencing, and what it is good for:

A

Sequence genes one cell/chromosome at a time (individually) - detailed info on most abundant microbes

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7
Q

Where are bacterial genes found?

A 
In chromosomes (1 or 2) in cytoplasm
Can have extra DNA in plasmids
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8
Q

___ ____ ____ are genes that can be transferred between bacteria

A

Mobile genetic elements

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9
Q

Average size of bacteria chromosomes:

A

5 million BP

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10
Q

Significance of the 16S gene:

A
  1. essential for bacteria cell function, so every bacteria must have at least 1
  2. 9 hypervariable sections (each bacteria type is unique) - acts as a “barcode” for identifying bacteria
  3. cannot be transferred horizontally (slow evolution makes tracing accurate)
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11
Q

What is the 16S gene useful for, in microbiology?

A
  1. phylogenic identification

2. evolutionary biology (“molecular clock” - estimate when species diverged/mutated)

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12
Q

The use of 16S Targeted amplicon sequencing combined with ____ _____ sequencing allows for rapid identification of most microbes in a microbiome. However, it does not tell us:

A

massive parallel

does not tell us how they interact

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13
Q

True/False; 16S targeted amplicon sequencing has been the most heavily used in microbiome studies

A

True

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14
Q

True/False: 16S rRNA sequencing can be applicable for all microbes in the human microbiome

A

False; only occurs in PROKARYOTES (not eukaryotes)

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15
Q

Why is 16S rRNA the gene used for microbiome studies (and not some other essential gene?)

A

Slow evolution (no horizontal gene transfer) -> more accurate taxonomy identification

Comprehensive databases and reliable info already exist

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16
Q

Limitations/problems with 16S rRNA sequencing:

A
  1. bacteria can have >1 16S rRNA gene
  2. only 30 out of 1300 phyla are formally described
  3. short sequences not reliable
  4. will take 1000+ years to sequence all remaining species
17
Q

How many base pairs are usually sequenced for 16S rRNA?

A

250

18
Q

16S rRNA sequencing is great for: (2) but not good for (3)

A

good:

  1. comparing bacterial populations (diff. body parts, states, organisms)
  2. determine changes in population from event/over time

bad:

  1. examine changes at species level
  2. elucidate functional potential of microbiome
  3. functional potential of individual microbes
19
Q

Describe the general process of metagenomics:

What does it tell you?

A

extract DNA from community -> cut up into small portions -> sequence everything -> try to piece it together

information about functions taking place; doesn’t tell you who does what

20
Q

What is PICRUSt? What is it used for, and when can’t it be used?

A

Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (software)

estimate of genes present in microbiome based on 16srRNA sequencing

Not good for poorly studied environments

21
Q

Which is more expensive, 16sr rRNA sequencing or metagenomic sequencing?

A

metagenomics (500-2500$)

22
Q

sequencing methods and software for 16s rRNA sequencing:

A

sequencing: MiSeq, HiSeq, MinION
processing software: QIIME, Mothur, RD Pipeline
interpretation software: R, microbiome analyst

23
Q

How is metagenomic data analyzed?

A

sequenced with HiSeq:

De novo assembly
Mapping to known genome
Mapping to functional gene databases

24
Q

Things to be critical of in sequencing studies:

A
  1. poor sequencing strategy
  2. confounded sample collection
  3. DNA extraction
  4. Data processing
  5. Small sample sizes
25
Q

How does the choice of primers affect the conclusion of 16S rRNA analysis?

A

need to pick primers for 5’ end and 3’ end (going both ways; forward and reverse); sequences should OVERLAP a fair amount to correct for error

(no overlap -> no correction -> small change in sequence can indicate totally different organism or operational taxonomic unit)

26
Q

What is a good set of primers to use?

A

548F, 806R

27
Q

What is the problem of “confounded data?”

A

other variables not accounted for that will affect the relationship between the two variables investigated; cannot establish a causal relationship between those two factors
(ex: not clear whether differences in microbiome of autistic children vs normal kids is due to autism, or due to their higher sugar diet)

28
Q

How does DNA extraction affect results of a study?

A

many methods available; changed method may give different results (extracts slightly different population)

29
Q

Why are meta studies concerning the microbiome less reliable?

A

Doesn’t consider differences in DNA extraction methods; each will have different microbes included/excluded

30
Q

What advantage does Mothur have over QIIME in regards to considering the reliability of a study?

A

Mothur will only read sequences that almost fully overlap (correct for error), so sequencing has to be done well
QIIME will produce result even for sequence with a lot of errors

31
Q

If you have chosen 2 primers to use that do not overlap very well, what data processing software do you use?

A

QIIME

32
Q

True/False: Autoclaving is sufficient to clean equipment for sequencing

A

False; autoclaving does not destroy DNA!

33
Q

What types of controls can be used?

A

negative control
generous donor sample
mock community

34
Q

What is alpha and beta diversity?

A

alpha: within samples
beta: between samples (phylogeny or non-phylogeny based)

Food Microbiology (21 decks)

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