ENCODE and TraDIS

Question 1

What was the ENCODE project?

(Incl its aims)

Answer 1

ENCODE = Encyclopaedia of DNA elements
Large international collaboration with the aim of identifying all the functional regions of the human genome

Question 2

What methods were used in the ENCODE project?

Answer 2

-5C (long range chromosomal interactions)
-ChIP seq (prot binding sites on genome)
-Whole genome bisulfide seq (WGBS) and reduced representation bisulfide seq (RRBS) (gene locations and expression)
-RNA-seq (gene expression/transcription levels)
-DNase-seq (euchromatin/transcriptional activity)

Question 3

What was the controversy around the ENCODE project?

Answer 3

-Disagreements over their definition of function elements in human genome
-they included every transcribed region (very liberal definition)
-their initial finding was that 80% of the human genome
-if only evolutionarily conserved elements were considered functional, would be 8.2% (but that wouldn’t include loci assoc with individual appearances)
-% depends where evolutionary evidence or genetic evidence is considered

Question 4

What is TraDIS?

Answer 4

TraDIS = Transposon directed insertion-site sequencing
-Sequencing technique where transposase is used to insert a desired gene (selective marker eg. antibiotic resistance) flanked by inverted repeat sequences

Question 5

How can DNA sequencing be used to determine the site of a transposon insertion?

Answer 5

High throughput sequencing (next gen and third gen seq techs) enabled identification of where transposon was inserted in TraDIS by mapping to reference genome

Question 6

How is transposon mutagenesis and TraDIS used to identify essential bacterial genes and genes important for infection?

Answer 6

-if tn interupts a gene, it will be inactivated
-if a gene is disrupted, mutant won’t survive
-genes without insertions are likely to be essential -> subgenic resolution important!