1. Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun 2015-5-21Group 4 1 Genomics and Epigenomics

2. Outline A powerful forward genetic biotechnology for phenotype related genes identification, genome annotation…… Backgrounds Biotechnologies Results Discussions 2015-5-21Wang Ting 2 (1) (2)

3. Background The ability to remove or inactivate single genes in cells is revolutionary; Insertion mutagenesis in a haploid background can disrupt gene function, using retroviral gene-trap vector to generate insertions (Jan E. Carette et al. Science. 2009) 2015-5-21Wang Ting 3  Extend by applying phenotypic interrogation via tag sequencing (PhITSeq) to examine millions of mutant alleles through selection and parallel sequencing

4. Backgrounds (Authors intro.) Jan E Carette: –A postdoc in the Brummelkamp lab –Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA. Papers: –Haploid genetic screens in human cells identify host factors used by pathogens. Science, November 27, 2009. –Ebola virus entry requires the cholesterol transporter Niemann- Pick C1. Nature, online on August 24, 2011. 2015-5-21Li Jun 4

5. Retrovirus A retrovirus is an RNA virus that is duplicated in a host cell using the reverse transcriptase enzyme to produce DNA from its RNA genome. The DNA is then incorporated into the host's genome by an integrase enzyme. The virus thereafter replicates as part of the host cell's DNA. Retroviruses are enveloped viruses that belong to the viral family Retroviridae. 2015-5-21Group 4 5 From google picture

6. Gene-trap insertion mutagenesis 2015-5-21Li Jun 6 International Gene Trap Consortium (IGTC) http://www.genetrap.org/tutorials/overview.html

7. Phenotype selection CDTs for phenotype selection Identify host factors required for the effects of backterial toxins; to determine whether CDTs of diverse origin and structure use some common or different factors for their entry and intoxication; 2015-5-21Li Jun 7

8. PhITSeq 2015-5-21Jiang Bai 8 Processing –Insertional mutagenesis -> Phenotypic selection -> sequencing -> Bowtie mapping to get insertion sites

9. Sequencing for selected population 2015-5-21Jiang Bai 9 Short DNA sequences flanking the inserted gene-trap vectors were amplified.

10. Results PhITSeq screens performed with CDTs secreted by different bacteria 2015-5-21Qin Zhiyi 10

11. Results (cont.) Gene-trap insertions identified in loci essential for CDT intoxication 2015-5-21Qin Zhiyi 11

12. Results (cont.) Loci linked to 12 separate phenotypes 2015-5-21Qin Zhiyi 12

13. Discussions advantages –Haploid cell line  powerful global gene disruption; –High throughput deep sequencing  analyze pools of cells, get genome-wide overviews of genes and enable rapid assessment of the spectrum of genes, assigning genes to phenotypes with high saturation and accuracy; –many phenotypes are accessible  efficient for the genome annotation, or comparative analyses; 2015-5-21Wang Ting 13

14. Discussions (cont.) disadvantages –Rely on the use of one particular human near-haploid cancer cell line (gene function is condition-specific); –compared to RNAi-based screens (can be applied to many cell types, but cannot achieve global gene disruption); –Genetic redundancy or interaction among mutant alleles may affect the selection and statistical results; 2015-5-21Wang Ting 14

15. The end Questions? Thanks! 2015-5-21Group 4 15