Functional Genomics Research Stream. Research Meeting: June 5, 2012 Summer Goals

Transcription

1 Functional Genomics Research Stream Research Meeting: June 5, 2012 Summer Goals

2 Meetings Tuesdays - NHB June 5 through July 24 Required 3:00pm to 4:30pm Alternate - TBA

3 Expectations Respect hourly commitment Be productive Own your project Be courteous to each other Be conscientious of our lab space Report your results on Results Central

4 Logistics Hours (9am - 6pm) With partner after hours Avoid flames and phenol after hours Key Access Cleaning Resource Scheduling

5 Goals Learning to analyze scientific publications Practicing RT-qPCR for gene expression profiling Reaching RNA-seq stage for two more experiments Utilization of gene knockout mutant yeast strains Finishing analysis of heat shock RNA-seq data and making draft of publication

6 Updates Dr. White vs. Travis Mentors - reduced hours High school students in lab Media protocol updated to include complete minimal and minimal Primer sequences (but not locations) have been updated

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9 30C 37C +24 hours Saccharomyces bayanus Saccharomyces cerevisiae Saccharomyces mikatae Saccharomyces paradoxus +48 hours 30C 37C 30C 30C 48 to 120 hours

10 Choose timepoint with largest expression change Analyze in 4 species of yeast Cold stress MMS-induced DNA damage UV Stress Restricted wavelength light Alkaline stress

11 S. everything RNA-seq Experiment experimental X stress reference normal growth

12 Use PCR to build DNA fragment you wish to incorporate into the genome. This fragment must contain a selectable marker. UPTAG primer PCR KanMX4 produces DOWNTAG primer KanMX4

13 4. Allow cells to incubate with fragment - homologous recombination occurs. KanMX4 ATG stop CHR III Gene X yeast cell

14 ESR genes Msn2/Msn4p. Wild-type, msn2 msn4, From Gasch et al. 2000

15 Yeast Resources Saccharomyces Genome Database yeastgenome.org gene information The Broad Institute annotation/fungi/comp_yeasts/ downloads.html ORF annotations used for RNA-seq analysis of 4 yeast species using bwa

16 Yeast Resources Scannell & Zill Publication high-quality, well-annotated, assembled genomes for 4 yeast species may use these references for the RNAseq alignment program tophat and differential expression analysis by cufflinks

17 PCR Verification with Positive & Negative Controls Gene X required gene Y chromosome pa pb pc pd +F +R ~500 bp ~500 bp ~200 bp PCR product kanmx4 required gene Y chromosome pa pkanb pkanc pd +F +R ~500 bp ~500 bp ~200 bp

18 Assignment 1 Download ApE (A plasmid editor) wayned/ape/ use yeast genome resources to design a primer set (pa and pb; or pc and pd) for gene deletion confirmation design one set by eye using ApE, another set using a design program (either ApE or the one linked on the course website)

19 What Do We Do with These Sequences? Well, we want gene expression information. REFERENCE GENOME Gene X TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATG read 1: GTTATCCACAGAATCAGGGGATAACG read 2: TTTGGTTGATGCGAGTGATTTTGATG read 3: read 4: read 5: read 6: CGTTATCCCCTGATTCTGTGGATAAC CATGGCAAAGGTAGCGTTGCCAATGA CCTGATGATGCATGGTTACTCACCAC TTAACCTGCATTAATGAATCGGCCAA

20 Count the Number of Reads (sequences) and Which Genes They Align To Gene A Gene B Gene C Gene D Gene E Control Exp Total Normalized Reads/ Total Gene Expression Control Exp. Exp./ Control /0.2 = /0.2 = /0.2 = /0.2 = /0.2 =2

21 Count the Number of Reads (sequences) and Which Genes They Align To Gene A Gene B Gene C Gene D Gene E Gene Expression Exp./ Control 0.1/0.2 = /0.2 = /0.2 = /0.2 = /0.2 =2 Gene Expression log2 (Exp./ Control) log2 (0.5) = -1 log2 (1) = 0 log2 (0.25) = -2 log2 (1.25) = 0.32 log2 (2) = 1

22 RNA-seq Analysis Align short RNA-seq reads to reference genome - count genes (using bwa, tophat,...) Get gene expression information (using Excel, cufflinks,...) Cluster genes with similar expression patterns using Cluster 3.0 (or another program) Visualize Cluster output using Java Tree View Analyze gene ontology of clustered genes Analyze DNA sequence motifs to explain gene expression patterns (using Meme,...)

23 What to Do for Next Week Assignment I Read Kvitek, Will and Gasch, see literature section of course website Look for guideline questions on Results Central soon Update your current progress on Results Central

24 Current Heat Shock Data I will send everyone the current heat shock data Take a look at it (if interested) using programs mentioned above Think about describing data in a similar context as the Kvitek, Will and Gasch, 2008 paper