6 TnSeq And ChIP Seq

Question 1

What is TIS

Answer 1

Transposon insertion sequencing

Have four variations to the method : we look at Tn-seq

The methods vary in their their preparation of the library’s but the genetic manipulations (like mutant generation) and data analysis are similar

Question 2

What is Tn-seq

Whag is the workflow of TIS (Tn-seq)

Answer 2

Insertion sequencing

1. Create random mutants:

• by having the transposon insert the abR (antibiotic resistance gene) randomly into the genome of the organism you want to analyze
• this make a mutant library with antibiotic resistance

2. Select and pool mutants:

• grow on plate with antibiotic resistance
• extract the DNA from cells that survived (one with the mutant genomes)
• making a transposon library

3. Fragment, add adaptors, and PCR amplify:

• fragment all the DNA for the pool of cells that survived (so that a transposon in each fragment)
• add adaptors to the ends of the dna
• PCR using one primer to target transposon and one to target the adaptor (so arounf the transposon)
• this amplifies the insertion sites of the transposon in all the genomes of the mutants you made

Sequencing and mapping:
- sequence all the amplicons
- map each read to a reference and determine the insertion sites of the transposons across the genome

Question 3

What’s the point of tn-seq

Answer 3

Can define what the minimal set of genes are needed to

Added more in other

Question 4

How do you analyze the data for tn-seq / TIS

Answer 4

Ex. Have a control (not treated with antibiotic) and a treated (treated with antibiotic) to see what genes might be involved in resistance

Differences in the presence of transposon in certain regions, show that some are more likely mutated than others, meaning there have diff function in those diff conditions.

Ex. Looking to tie genes back to antibiotic resistance

Gene involved in sensitivity:
- if the gene for sensitivity is high in the treated low in control , this means it is more likely to be mutated in the treated because those ones that survive wouldn’t have a function sensitivity gene
- that’s how you know that gene is for sensitivity

Gene involved in resistance:
- if the gene is for resistance to antibiotic, the treated sample wouldn’t have reads because the gene is less likely to be mutated in this condition so the cells can survive, the untreated has more reads because it is okay to be mutated
- that’s how you know it’s for resistance

Essential gene:
- If a gene is essential you would see no transposon reads for it in the both the control and treated.
- this is because less transposon reads under many conditions means the gene is less likely to be mutated, meaning it’s essential.
- that’s how you know it’s essential for growth because not mutated in either condition

Gene has not essential for growth
- see lot of reads because it can have lots of mutations
- that gene is dispensable

Question 5

What are the applications of TIS

Answer 5

1. Motility TIS:
   - want to see the genes that are important for motility of bacteria
   - do swim assay with the mutants where they swim out in diff directions at diff rates
   - sequence the ones the travels furthest and the least and compare the genes that were mutated in the two (to see which genes essential for motility and not)
2. Density gradient:
   - Bactria can have diff cell density
   - sequencing mutants that travelled diff in density gradient and see what genes essential for more or less density
3. TraDIsort
   - sorting the mutants based on their fluoresce of a fluorescent drug analog etBr
   - more fluorescence means more drug uptake
   - sort the ones that are less Fluor and highest flour and compare the sequences to see which genes essential for drug efflux
4. dTN sequencing: droplet
   - encapsulate mutant bacteria in droplets of media
   - can count number of cells present in a droplet to find their growth and sequence to see genes essential for growth

Question 6

What is chip-seq

Answer 6

Chromatin immunoprecipitation sequencing

Used to collect and enrich DNA fragments that interact with a specific tagged protien

To see what dna interacts with that protien and defines the protien dna binding sites using multiple sequencing alignment

Question 7

Key issues of chip-seq

Answer 7

You have to make sure the epitope tagged protien is still functional before doing the experiment

Question 8

How does a chipseq data analysis work

Answer 8

Use a z test to look for gDNA fragments that are over represented in a library relative to all the others (those are the ones that bind the protien)

Usually correlated with data from transcriptomics:
- take rna seq data with this data and reveal which genes in a cell are directly modified by the protien and consequently differentially expressed

The key finding are verified using additional molecular assays like emsa

Question 9

Chipseq workflow

Answer 9

1. DNA cross linked with it’s interacting protiens using crosslinker
2. Enzyme shears the dna , antibody bind to the target protien
3. Bound DNA is uncrosslinked and purified
4. Sequence the DNA and look for regions of enrichment compared to reference genome to see what was captured

Question 10

Explain OMPR chip seq

Answer 10

Can see which fragments of the ompR regulon are over represented after chipseq and those are sequencing that bind the OmpR

Can also do differential expression analysis to see if genes in mutant regulon don’t bind ompR

Can also show gene function

Question 11

Explain EMSA

Answer 11

Electrophoretic mobility shift assay

A Way to check that chipseq data is good

1. Protien purified in vitro mixed with fluorescent labeled dna seqeunce you think it binds to
2. Protien binding to that sequencing makes the band shift up (because higher MW)

Can mutate that protien and show that it’s essential for binding by doing EMSA again