Biol 456/656 Molecular Epigenetics Lecture #2 Wed August 26, 2015.

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Presentation transcript:

Biol 456/656 Molecular Epigenetics Lecture #2 Wed August 26, 2015

5 BIG IDEAS 1. Instead of measuring your favorite gene’s expression levels, measure everything! RNA-seq CHIP-seq Bisulfite Sequencing Gro-seq 3’-seq Ribosome profiling

5 BIG IDEAS 2. RNA: more than a messenger! lncRNA piRNA siRNA circRNA microRNA snoRNA

5 BIG IDEAS 3. Gene Regulation: From bench to

5 BIG IDEAS (covered in 456/656) 4. Lineage Reprogramming

5 BIG IDEAS 5. Breaking the Genome with CRISPR/Cas

Gene expression. Transcription basics Eric Lander, MIT REVIEW OF THE BASICS

Gene-centric viewGenome-wide view Biomedical science is undergoing a rapid change

Technology changes everything

Motorola DynaTAC 8000X $ Motorola Moto G $

Sanger Sequencing -400 bases 1977 Illumina Hiseq billion reads 2014

$10,000,000 human genome 2008 $1000 human genome sequencing-system/system.html

--$100 million --$10,000 --$1 million Cost of sequencing a human genome Moore’s Law for computing costs Hayden, Nature 2014 Computing power per $ ~doubles every year

Methods to measure 1 RNA at a time – Northern Blot – RNAse protection assay – Quantitative RT-PCR

PCR Reverse Transcription -create cDNA from RNA REVIEW OF THE BASICS

Quantitative PCR End Point PCR REVIEW OF THE BASICS

mRNA from cells/tissues Fragmentation Convert to cDNA Adapter ligation RNA-seq: quantify the transcriptome

cDNA/DNA Single molecule array Sample preparation Cluster growth 5’ 3’ G T C A G T C A G T C A C A G T C A T C A C C T A G C G T A G T Image acquisition Base calling T G C T A C G A T … Sequencing Sequencing by synthesis

\ 100 Microns Sequencing 100’s Million Clusters Per Flow Cell

RNA-seq quantitative gene expression novel transcript discovery

RNA-seq data from Fly tissues Visualized using Integrated Genomics Viewer (IGV) 1 read “Stranded” Mei-P26 OVARY

How do you deal with so much data? Different skill set required – Non GUI (Graphical User Interface) software – Unix – Various programming languages (Perl, Python) – Statistics/Visualization platform (R) – Visualize sequencing data (IGV, UCSC genome browser)

chr3R:18,842,756-18,864,455 bin/hgTracks?hgS_doOtherUser=submit&hgS_otherUserName=pedstunite& hgS_otherUserSessionName=brent RNA-seq track of 6-8 hr embryos

Become a hybrid scientist! -alternatively become a “genome-informed” health care worker