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Genomics and Epigenomics
Group 4 members: Wang Ting, Jiang Bai, Qin Zhiyi, Li Jun
2017/5/23
Group 4
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Outline
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 A powerful forward genetic biotechnology for
phenotype related genes identification, genome
annotation……
• Backgrounds
• Biotechnologies
• Results
• Discussions
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Wang Ting
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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)
 Extend by applying
phenotypic interrogation
via tag sequencing
(PhITSeq) to examine
millions of mutant alleles
through selection and
parallel sequencing
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Wang Ting
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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 NiemannPick C1. Nature, online on August 24, 2011.
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Li Jun
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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.
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Group 4
From google picture
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Gene-trap insertion mutagenesis
International Gene Trap Consortium (IGTC)
http://www.genetrap.org/tutorials/overview.html
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Li Jun
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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;
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Li Jun
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PhITSeq
• Processing
– Insertional mutagenesis -> Phenotypic selection -> sequencing ->
Bowtie mapping to get insertion sites
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Jiang Bai
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Sequencing for selected population
Short DNA sequences flanking the
inserted gene-trap vectors were amplified.
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Jiang Bai
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Results
• PhITSeq screens performed with CDTs secreted by different
bacteria
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Qin Zhiyi
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Results (cont.)
• Gene-trap insertions identified in loci essential for CDT
intoxication
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Qin Zhiyi
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Results (cont.)
• Loci linked to 12 separate phenotypes
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Qin Zhiyi
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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;
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Wang Ting
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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;
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Wang Ting
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The end
• Questions?
• Thanks!
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Group 4
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