10 - Techniques in Microbiome Studies

Question 1

What is the microbiome

Answer 1

the combined genetic material of the organisms found in a particular environment

Question 2

what is the microbiota

Answer 2

the community of microorganisms found in a particular environment

Question 3

What are the techniques used to study microbiomes?

Answer 3

1. 16s rRNA targeted amplicon sequencing
2. Metagenomics
3. Single-cell genomics/ read cloud sequencing

Question 4

What information is given by the 16srRNA genes sequencing

Answer 4

give information at which bacteria are inhabiting an environment

Question 5

What information is given by the metagenomics?

Answer 5

gives information on the functional properties of a community because it gives all the genetic material from a environment

Question 6

What information is given by the single-cell genomics ?

Answer 6

give detailed information about the genetic information of the most abundant organism of an environment

Question 7

Why is the 16s rRNA is used in microbiome studies

Answer 7

1-because every bacteria has at least one 16s rRNA
2- because this gene is like a barcode telling you the identity of the bacteria
3- gene only present in the procaryotes and not in eukaryotes
4- slow evolution in that sequence
5- contains 9 consitent regions so it can be used to make a primer

Question 8

How long is the sRNA gene ?

Answer 8

1542 base pair

Question 9

T or F : the 16 sRNA is present in eukaryotes

Answer 9

false

Question 10

What are the limitation of using the 16s RNA targeted amplicon sequencing? (5)

Answer 10

1- some bacteria have more than one 16s rRNA gene and everybody is ignoring this
2- we only know a small percentage of what is out there
3- not accurate to determine the richness of an environment because of the misreading of the sequence
4- not all the bacterial species have been classified
5- 16s rRNA is only a short portion of the whole genome of the bacteria and you sequence only 250 bp

Question 11

T or F : most bacteria have 2 chromosome

Answer 11

false most have 1 chromosomes and multiple plasmid

Question 12

When is it wrong using the 16s rRNA sequencing?

Answer 12

when you want to know what a microbiome is capable of doing, you need to use metagnomics

Question 13

why don’t we use metagenomics all the time?

Answer 13

because much more expensive than 16s rRNA

Question 14

What is PICRUSt

Answer 14

Provides an estimate of the genes present in the metagenome based on 16S rRNA targeted amplicon sequencing

Question 15

T or F :

Answer 15

PICRUSt is much more accurate samples from the Human Microbiome Project than other microbial samples

Question 16

What should we be critical when looking at a study using the 16s rRNA sequencing

Answer 16

1- poor sequencing strategy
2- confounded sample collection
3- DNA extraction 
4- Data processing 
5- small sample size

Question 17

What is the best number of bp you should use for sequencing ?

Answer 17

250 bp

Question 18

bead beating will help you extract the gram ____ but might shear the gram ___

Answer 18

helps extract the gram +

might shear the gram -

Question 19

What is the problem with DNA extraction

Answer 19

the various kits will extract a slightly different population of cells

Question 20

Why should you be critical of meta studies of the microbiome

Answer 20

because of the differences in the sample collection, DNA extraction, data processing, sample size and the sequencing strategy also

Question 21

What are the different data processing software?

Answer 21

• mothur

- QIIME

Question 22

which software is this : Does not require that reads fully overlap and is recommended if you choose primers that don’t fully overlap forward and reverse reads

Answer 22

QIIME

Question 23

which software is this : will not process sequence reads that don’t (almost) fully overlap

Answer 23

mothur

Question 24

What is wrong of not using a control?

Answer 24

there is DNA everywhere, in the kit, on the pipettes and on a lot of things

Question 25

Does autoclaving removes DNA?

Answer 25

no

Question 26

What is the alpha diversity?

Answer 26

diversity in a class, within samples

Question 27

what is the beta diversity ?

Answer 27

diversity between the sample example the diversity of all the classes of mcgill

Question 28

T or F : seeing that the authors have used mothur means that the sequencing was done well

Answer 28

true, because the program can’t process if the sequencing wasn’t done well