BIOL335: Functional genomics

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Information about BIOL335: Functional genomics
Science

Published on October 1, 2014

Author: ppgardne

Source: slideshare.net

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Course material for:
http://www.canterbury.ac.nz/courseinfo/GetCourseDetails.aspx?course=BIOL335

Functional Annotation of Genomes Paul Gardner September 26, 2014 Paul Gardner Functional Annotation of Genomes

Discussion I How can we identify functional regions within genomes? Paul Gardner Functional Annotation of Genomes

Loss-of-function methods: knock-outs & knock-downs I Two approaches for testing gene function: I Knocking out genes by deletion or disruption of functional regions I Knocking down genes by introducing antisense RNA (i.e. RNAi) I Assay the impact of the knock out/down Paul Gardner Functional Annotation of Genomes

Can we knock genes out on a large scale? I Idea 1: generate populations of mutants with transposable elements. Hutchison III et al. (1999) Global Transposon Mutagenesis and a Minimal Mycoplasma Genome. Science Paul Gardner Functional Annotation of Genomes

Tn-seq I Transposons are represented by pink lines, sequencing adaptors by blue, genomic DNA by black and PCR primers by green. Barquist, Boinett & Cain (2013) Approaches to querying bacterial genomes with transposon-insertion sequencing. RNA Biology Paul Gardner Functional Annotation of Genomes

Tn-seq (called TraDIS in the UK) I Electrotransformation with Tn5-derived transposon/transposase complex containing a kanamycin- resistance gene I > 10 transformations per batch at 42; 000 146; 000 mutants per batch; 13 batches ! 1.1 million mutants I Short read sequencing with transposon speci

c primers Langridge et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants Genome research Paul Gardner Functional Annotation of Genomes

Tn-seq Langridge et al. (2009) Simultaneous assay of every Salmonella Typhi gene using one million transposon mutants Genome research Paul Gardner Functional Annotation of Genomes

Tn-seq on RNA in Salmonella 35 30 25 20 15 10 5 0 RNAseq reads 100 80 60 40 20 0 TraDIS reads 994000 994100 994200 994300 994400 genome coordinate dsrA putative transcript yodD Paul Gardner Functional Annotation of Genomes

Comparative Tn-seq Barquist L, Langridge GC, Turner DJ, Phan MD, Turner AK, Bateman A, Parkhill J, Wain J, Gardner PP (2013) A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. Nucleic Acids Research. Paul Gardner Functional Annotation of Genomes

Comparative Tn-seq (A) the overlap of all genes (outer circles) and required genes (inner circles) Black numbers refer to all genes, white numbers to required genes. (B) the overlap of all required genes between S. Typhimurium, S. Typhi and E. coli. Barquist L, Langridge GC, Turner DJ, Phan MD, Turner AK, Bateman A, Parkhill J, Wain J, Gardner PP (2013) A comparison of dense transposon insertion libraries in the Salmonella serovars Typhi and Typhimurium. Nucleic Acids Research. Paul Gardner Functional Annotation of Genomes

Some issues... Paul Gardner Functional Annotation of Genomes

What's more useful to know, essential or functional? I How could these be distinguished? I Are essential genes guaranteed to be functional? Are functional genes essential? Paul Gardner Functional Annotation of Genomes

Knockouts in Eukaryotes: mouse KOs I Idea 2: systematic KOs at targeted loci. I Targeted 9,000 ( 45%) genes Skarnes et al. (2011) A conditional knockout resource for the genome-wide study of mouse gene function. Nature. Paul Gardner Functional Annotation of Genomes

Knockout mouse production I Replace target gene, using homologous recombination, with a neomycin resistance gene Image source: https://en.wikipedia.org/wiki/Knockout mouse Paul Gardner Functional Annotation of Genomes

Knockout mouse

ndings Lewis et al. (2009) An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nature Genetics. Mencia et al. (2009) Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss. Nature Genetics. Paul Gardner Functional Annotation of Genomes

miR-96 and deafness 5’ 3’ 0 1 Sequence conservation U G G A G C C S A U U U U G G C A C U A G C A C A U U U U U G G M A R U A U A A C C G U G A Y G U G U A A C U A C G C U U S U G U C U C U U U C C A C G C G Lewis et al. (2009) An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice. Nature Genetics. Paul Gardner Functional Annotation of Genomes

Some issues... Paul Gardner Functional Annotation of Genomes

Knockouts in Eukaryotes: cool kids now use CRISPR/CAS I Idea 3: systematic KOs at targeted loci using CRISPR/CAS. I Targeted 18,080 ( 90%) genes with 64,751 unique guide sequences I Identify genes essential for cell viability in cancer and pluripotent stem cells Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science. Cong et al. (2013) Multiplex Genome Engineering Using CRISPR/Cas Systems. Science. Mali et al. (2013) RNA-Guided Human Genome Engineering via Cas9. Science. Paul Gardner Functional Annotation of Genomes

Knockouts in Eukaryotes: CRISPR/CAS Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science. Paul Gardner Functional Annotation of Genomes

Knockouts in Eukaryotes: CRISPR/CAS - melanoma Shalem et al. (2014) Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Science. Paul Gardner Functional Annotation of Genomes

Knockdowns I What about RNA? I Idea 4: systematic, genome-wide RNAi screens. Paul Gardner Functional Annotation of Genomes

Knockdowns in Eukaryotes: worms I  86% of the 19,427 C. elegans genes knocked down I Assayed larval lethality, slow growth & defects I Phenotypes for 1,722 genes, 2 3 of these were novel Kamath et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Science. Paul Gardner Functional Annotation of Genomes

Knockdowns in Eukaryotes: worms I NonV - nonviable (incl. sterility) I Gro - growth defects I Vpep - viable post-embryonic phenotype Kamath et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Science. Paul Gardner Functional Annotation of Genomes

Knockdowns in Eukaryotes: worms Nonv Gro Vpep Kamath et al. (2003) Systematic functional analysis of the Caenorhabditis elegans genome using RNAi. Science. Paul Gardner Functional Annotation of Genomes

Knockdowns in Eukaryotes: human & mouse cell lines I 5,000 unique shRNA-expressing lentiviruses that target 1,028 ( 5%) human genes I  100 candidate regulators of mitotic progression and proliferation Mofatt et al. (2006) A Lentiviral RNAi Library for Human and Mouse Genes Applied to an Arrayed Viral High-Content Screen. Cell. Paul Gardner Functional Annotation of Genomes

Knockdowns in Eukaryotes: human & mouse cell lines I designed 700 unique shRNA-expressing lentiviruses that target 133 ( 0:7%) human genes that have been associated with breast cancer Possemato et al. (2011) Functional genomics reveal that the serine synthesis pathway is essential in breast cancer. Nature. Paul Gardner Functional Annotation of Genomes

Some conclusions I Loss of function methods are a powerful tool for identifying protein function I The technology is evolving rapidly I Screening large numbers of phenotypes is now a major bottleneck & consistently linking these datasets is an open problem I A useful independent dimension to traditional" measures of gene function e.g. evolutionary conservation and expression Paul Gardner Functional Annotation of Genomes

Important dates Week 10: Tue 30 Sep 14:00 16:00 BIOL335 COM.LAB - Erskine 248 - FREE Thu 02 Oct 12:00 13:00 BIOL335 LECTURE - Erskine 031 - FREE Fri 03 Oct 13:00 14:00 BIOL335 LECTURE - Erskine 031 - FREE Week 11: Tue 07 Oct 14:00 16:00 BIOL335 COM.LAB - Erskine 248 - PG: MOCK PRACTICAL TEST. Thu 09 Oct 12:00 13:00 BIOL335 LECTURE - Erskine 031 - FREE Fri 10 Oct 13:00 14:00 BIOL335 LECTURE - Erskine 031 - FREE Week 12: Tue 14 Oct 14:00 16:00 BIOL335 COM.LAB - Erskine 248 - PG: PRACTICAL TEST (20%) Thu 16 Oct 12:00 13:00 BIOL335 LECTURE - Erskine 031 - Tutorial: prep for exam Fri 17 Oct 13:00 14:00 BIOL335 LECTURE - Erskine 031 - Tutorial: prep for exam Fri 17 Oct Lectures End for Semester 2 Thu 30 Oct 14:30 Final Exam: (40%) Paul Gardner Functional Annotation of Genomes

Genome annotation review I Genome annotation overview of methods I Sequence alignment I Homology search I Genetic selection I Functional genomics Paul Gardner Functional Annotation of Genomes

The End Paul Gardner Functional Annotation of Genomes

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